TP53 PIN3 and MDM2 SNP309 polymorphisms as genetic modifiers in the Li-Fraumeni syndrome: impact on age at first diagnosis.

BACKGROUND: Li-Fraumeni and Li-Fraumeni-like syndromes (LFS/LFL), characterised by the development of multiple early onset cancers with heterogeneous tumour patterns, are associated with germline TP53 mutations. Polymorphisms in the TP53 pathway (TP53 PEX4 at codon 72, rs1042522; MDM2 SNP309, rs2279744) have modifier effects on germline TP53 mutations that may account for the individual and familial diversity of tumour patterns. METHODS AND RESULTS: Four polymorphisms were analysed in a series of 135 Brazilian LFS/LFL cancer patients (32 TP53 mutation carriers and 103 wild-type subjects). We report for the first time that another polymorphism in the TP53 gene, TP53 PIN3 (rs17878362), has a strong modifier effect on germline TP53 mutations. This polymorphism, which consists of a 16 bp duplication in intron 3 (A1, non-duplicated allele; A2, duplicated allele), is associated with a difference of 19.0 years in the mean age at the first diagnosis in TP53 mutation carriers (n = 25, A1A1: 28.0 years; n = 7, A1A2: 47.0 years; p = 0.01). In addition, cancer occurrence before the age of 35 years is exclusively observed in A1A1 homozygotes. In this series, the effect of TP53 PEX4 and MDM2 SNP309 on age at diagnosis was similar to the one reported in other series and was smaller than the one of TP53 PIN3 (TP53 PIN3: difference of 19.0 years; TP53 PEX4: 8.3 years; MDM2 SNP309: 12.5 years). CONCLUSION: These results suggest that TP53 PIN3 is another polymorphism in the TP53 pathway that may have a modifier effect on germline TP53 mutations and may contribute to the phenotypic diversity of germline TP53 mutations associated with LFS/LFL patients.

WITHDRAWN: Expression of p53, p63, and p73 isoforms in squamous cell carcinoma and adenocarcinoma of esophagus☆,☆☆

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Distinct pattern of TP53 mutations in squamous cell carcinoma of the esophagus in Iran.

Extremely high rates of squamous cell carcinoma of the esophagus (SCCE) are observed in Iran, reflecting unknown, genetic and/or epidemiological risk factors. Among genetic alterations in SCCE, TP53 mutations are the most frequent, vary among populations, and may provide clues on etiological mechanisms. We have analysed mutations in TP53 (exons 5-8) in 98 SCCE from Iran by temporal temperature gel electrophoresis and direct sequencing. We found 58 mutations in 49 patients (50%), with a high prevalence of C to T transitions at CpG dinucleotides (29.3%). The TP53 mutation pattern in Iran was significantly different from that observed in SCCEs from high incidence areas of China and Western Europe (P=0.007). Moreover, the prevalence of mutations at A : T base pairs (transitions and transversions) was higher in men than in women (38.7% vs 11.1%, P=0.033). COX-2 overexpression was detected in 69% of the cases evaluated (24/35), without significant association with TP53 mutation. Accumulation of nitrotyrosine, a marker of protein damage by excess levels of nitric oxide, was observed in tumor cells in six of 18 [corrected] cases analysed. These results are consistent with the hypothesis that several factors are involved in TP53 mutagenesis in Iran. These factors include a baseline of chronic inflammatory stress, which may have a multiplicative impact on the sensitivity of esophageal cells to exogenous factors of risk.

TP53 mutations, amplification of P63 and expression of cell cycle proteins in squamous cell carcinoma of the oesophagus from a low incidence area in Western Europe.

In Europe, high incidence rates of oesophageal squamous cell carcinoma (SCCE) are observed in western France (Normandy and Brittany) and in north-eastern Italy. Analysis of TP53 mutations in tumours from these regions has shown a high prevalence of mutations at A:T basepairs that may result from DNA damage caused by specific mutagens. However, the spectrum of TP53 mutations in regions of low incidence is unknown. We report here TP53 mutation analysis in 33 SCCE collected in Lyon, an area of low incidence. These tumours were also examined for MDM2 and P63 amplification, and for expression of p16(INK4a/CDKN2a), cyclin E, p27(Kipl)and Cox2. TP53 mutations were detected in 36% of the cases (12/33). In contrast with regions of high incidence, the mutation spectrum did not show a high prevalence of mutations at A:T base pairs. P63 was amplified in 5/32 cases tested (15.5%). No amplification of MDM2 was found. Expression studies revealed frequent loss of p16(INK4a/CDKN2a)(46%) and p27(Kipl)(25%) expression, and frequent overexpression of Cyclin E (70%) and Cox2 (42%). Overall, these results indicate that in Europe, SCCE from areas of high and low incidence present a similar pattern of molecular alterations but differ by the type of TP53 mutations.

Molecular and clinical differences between adenocarcinomas of the esophagus and of the gastric cardia.

Adenocarcinoma of the esophagus (ADCE) with Barrett's mucosa and adenocarcinoma of the cardia (ADCC) are often reported as a single pathological entity. In this study we have used strict anatomical-pathological criteria to distinguish between these two lesions and we have investigated their differences in TP53 mutations, MDM2 gene amplification, and cytokeratin expression. DNA was extracted from the tumor areas of formalin-fixed, paraffin-embedded sections in 26 ADCC and 28 ADCE patients. TP53 mutations were detected by temporal temperature gradient electrophoresis and identified by sequencing. MDM2 amplification was assessed by differential polymerase chain reaction. The expression of cytokeratins 4, 7, and 13 was examined by immunohistochemistry. In ADCC, the male to female ratio was 1.8:1, compared to 27:1 in ADCE. Five ADCC patients had a history of other neoplasms, compared to only one ADCE patient. The two types of tumor differed in the prevalence of TP53 mutations (31% in ADCC and 50% in ADCE) and of MDM2 gene amplification (19% in ADCC and 4% in ADCE), and in the pattern of expression of cytokeratin 7 (positive in 100% of ADCE and in 41% of ADCC) and cytokeratin 13 (positive in 81% of ADCE and in 36.5% of ADCC). ADCE and ADCC differ in their clinical characteristics, in the prevalence of TP53 mutations and MDM2 amplifications, and in the patterns of cytokeratin expression. These results support the notion that ADCC and ADCE are distinct pathological entities.

Abnormal expression of the ATM and TP53 genes in sporadic breast carcinomas.

The ataxia telangiectasia gene (ATM) has been implicated as a risk factor in the development of sporadic breast carcinomas. ATM protein expression was analyzed by immunohistochemistry in 17 breast carcinomas with two monoclonal antibodies whose immunohistochemical use was first validated by comparing the immunoreactivity observed in spleen samples from ataxia telangiectasia and trauma patients. In normal breast ducts, ATM showed nuclear expression in the epithelial but not in the myoepithelial cells. In contrast, this nuclear expression was absent or low in the epithelial cancer cells in 10 of 17 (59%) of the tumors studied. Allelic imbalance in the ATM gene was found in three of seven tumors examined. Two of these showed reduced ATM protein expression, but this did not correlate with the presence of ATM mutations in the tumor DNA detected by restriction endonuclease fingerprinting screening. These results suggest that the reduced ATM protein expression could be attributable, in certain tumors, to deletions or rearrangements within or close to the ATM gene. Positive p53 immunostaining was found in 10 tumors, with TP53 mutations detected in 8. Three tumors had both low ATM expression and mutated TP53. Our results indicate that in the majority (15 of 17) of the sporadic breast carcinomas examined, not only is the functionality of the ATM-p53-mediated DNA damage response compromised, but also other signaling pathways activated by these two multifunctional proteins are likely to be impaired, which could be a contributing factor to tumor development and progression.

TP53 mutations and MDM2 gene amplification in squamous-cell carcinomas of the esophagus in south Thailand.

Squamous-cell carcinoma of the esophagus (SCCE) shows geographic variations in incidence that are thought to reflect the etiological involvement of environmental or dietary risk factors. Mutations of TP53 are frequent in SCCE, and there is evidence that both the frequency and type of these mutations may differ from one geographic area to the other. Although SCCE is relatively rare in most parts of Thailand, the province of Songkhla (south Thailand) has been described as a high-risk area for SCCE. We have analyzed 56 SCCE cases from this area for TP53 mutations by denaturing gradient gel electrophoresis (DGGE, exons 5-8) and direct DNA sequencing. The same tumors were also analyzed for MDM2 gene amplification by differential PCR. TP53 mutations were detected in 23 cases (41%). In contrast, clear amplification of MDM2 was detected in only 2 cases (4%), both of which contained wild-type TP53. Comparison with published results from other geographic areas of high SCCE incidence revealed that the spectrum of TP53 mutations in south Thailand is similar to that observed in central China (Henan Province) but clearly differs from that of SCCE from western Europe (Normandy, France; northern Italy), with more G:T transversions and fewer mutations affecting A and T base pairs. These results suggest that SCCE from south Thailand and from central China may involve similar risk factors.

Establishment and characterization of a spontaneously immortalized myofibroblast cell line derived from a human liver angiosarcoma.

BACKGROUND/AIM: Fibrosis and/or cirrhosis are present in the precursor stages of most liver cancers. However, little is known about the reciprocal interactions of fibroblasts, mainly responsible for fibrosis, and the other liver cells. We report here the isolation of a new liver myofibroblast cell line from a human liver angiosarcoma and its characterization. METHODS: The cells were isolated by the explant technique and characterization was performed, on one hand, using immunohistochemical and ultrastructural analysis and, in the other hand, by determining their karyotype, ras and p53 status and their tumorigenic properties. RESULTS: To date, the cells have undergone approximately 170 population doublings and are still proliferating. Immunohistochemically, they were negative for desmin, smooth muscle myosin, cytokeratin 19 and von Willebrand factor, positive for vimentin and alpha-smooth muscle actin, with an important deposition of fibronectin around the cells. Ultrastructure showed particularly cytoplasmic microfilament bundles. Their chromosome number ranged from 38 to 168 with a bimodal population, near diploid and hypotetraploid. No mutations were found in codons 12, 13 or 61 of Ha-, Ki- and N-ras genes but a homozygous missense mutation in codon 179 (CAT-->CTT) was detected in the p53 gene. They were unable to form foci in soft agar or tumors in nude mice. CONCLUSIONS: Taken together, these results show that these cells, called BM 2.2.1, exhibited typical myofibroblast-like features. Although they contained a karyotype suggestive of tumoral cells and a homozygous mutated p53 gene, they were not tumorigenic. The nature of these cells and the abnormalities of the p53 gene and the karyotype, suggest that: i) they were a component of the tumor stroma, and ii) they could have been involved in angiosarcoma development. Thus, this cell line may be valuable for the study of cellular interactions in liver carcinogenesis.

Analysis of p53, p16MTS, p21WAF1 and H-ras in archived bladder tumours from workers exposed to aromatic amines.

Exposure to aromatic amines is considered a major risk factor for the development of bladder cancer. In this study, we have analysed the pattern of point mutations in several tumour genes in 21 cases of bladder cancer arising among western European workers exposed to aromatic amines in an attempt to determine whether this exposure may be associated with a unique spectrum of mutations. Of the four genes analysed (p53, p16MTS1, p21WAF1 and H-ras), only p53 showed a high frequency of mutations (in 8 out of 21 cases, 38%). Two mutations were found in p16, one in H-ras and none in p21 exon 3. All mutations were at G:C base pairs, mostly at non-CpG residues. This spectrum of mutations, which is highly suggestive of an involvement of exogenous carcinogens, is however identical to the spectrum of p53 mutations detected in bladder cancers of the general population. In exposed workers, p53 mutations were associated with tumour grade and with high occupational and tobacco exposure. Taken together, our data suggest that the same carcinogens may be responsible for the development of bladder cancers in workers exposed to aromatic amines and in the general population.

Inactivation of the p53 protein in cell lines derived from human esophageal cancers.

Alteration of the p53 gene is thought to be important in the early stages of human esophageal cancers, but how this confers a selective advantage to esophageal cancer cells is unknown. In this report, we analyzed 9 cell lines derived from human esophageal cancers (TE-1, TE-3, TE-6, TE-7, TE-9, TE-10, TE-11, TE-13 and TE-15) for mutations in the p53 sequence, p53 protein expression and p53 protein DNA-binding activity. The cell lines could be grouped in 3 categories, including (1) cell lines with mis-sense mutations in the coding sequence and accumulation of mutant proteins (TE-1, TE-6, TE-10 and TE-11); (2) cell lines expressing truncated forms of p53 as a result of frameshift (TE-9) or splice-site (TE-15) mutations; and (3) cell lines with wild-type p53 sequences but with impaired expression of p53 mRNA and protein, suggesting that p53 is inactivated by transcriptional repression (TE-3, TE-7 and TE-13). With the exception of TE-1, none of the cell lines exhibited p53-DNA-binding activity. In TE-1, a mutation at codon 272 (methionine to valine) generated a protein that retains basal DNA-binding activity, but that was not activated in response to DNA damage, suggesting that this mutation prevented p53 induction by genotoxic stress. Thus, p53 activity was impaired in all esophageal cell lines, including those containing wild-type p53 sequences.

Low frequency of p16/CDKN2 gene mutations in esophageal carcinomas.

Mutational analysis of the p16/CDKN2 gene was conducted by direct sequencing of the whole coding sequence (exons 1-3 and flanking splicing sites) in 21 esophageal squamous-cell carcinomas and 3 adenocarcinomas from a high-incidence area of Italy. Two inactivating mutations were found in exon 1 of the gene (both in squamous-cell carcinoma), whereas no mutations were detected in exon 2, where most of the sequence changes reported so far have been located, or in exon 3. Southern blot analysis of exon 2 in this set of samples and in a complementary set of 12 tumor samples from France did not show homozygous deletions or detectable gene rearrangements. Thus, p16/CDKN2 gene alterations do not appear to play a major role in the group of patients examined.

p53 mutations in esophageal tumors from high-incidence areas of China.

Carcinomas of the upper digestive tract (squamous-cell carcinoma of the esophagus, adenocarcinoma of the cardia) from 24 patients residing in Linxian (China) and near-by high-incidence areas were analyzed for mutations in exons 5-8 of the p53 tumor-suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 50% of the specimens. Eleven tumors harbored a single base-pair substitution leading to either an amino-acid substitution (8 tumors) or a chain-termination signal (3 tumors), and one tumor revealed a 15-bp deletion in exon 7 with a silent base substitution adjacent to the deletion site. Mutations occurred in all 4 exons examined, with a preponderance in exon 5. Of the 6 mutations identified among the 14 adenocarcinomas examined, 3 were G to T transversions, a mutation that has thus far been absent from reported mutations in Barrett's esophageal adenocarcinomas and dysplasias from patients residing in Europe and North America.

Screening for p53 gene mutations in archived tumors of workers occupationally exposed to carcinogens: examples from analysis of bladder tumors.

Point mutations in the p53 tumor suppressor gene are the most common genetic alterations in human cancers. The nature and location of these mutations can be informative in assessing the importance of putative carcinogenic agents. Potential associations between a given carcinogen and a specific mutation pattern can be substantiated when the exposure history of the patients is known. While the past exposure to environmental risk factors is often difficult to determine, documented occupational exposure to carcinogens presents a unique situation for evaluating this approach. Analysis usually involves working with paraffin-embedded tissues, fixed under conditions suboptimal for genetic analysis and stored for many years, since frozen tissues are not available in sufficient numbers. The particular methodological problems encountered with fixed samples are discussed here, using as illustration an ongoing study of oncogene and tumor suppressor gene mutations in archived bladder tumors of workers exposed to aromatic amines and nonexposed patients.

Assay by inhibition of repair to measure O6-methylguanine in DNA.

A procedure for measuring the level of O6-methylguanine (O6-meG) in DNA is described. Repair of 32P-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O6-meG. Each methylated DNA sample inhibited the repair of oligodeoxynucleotide substrate to an extent dependent upon O6-meG concentration. Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O6-meG concentration. We report the method of calculation of the O6-meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs. This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used. The method requires only 0.1-10 micrograms of DNA, with a limit of detection of 0.8 fmol of O6-meG per microgram of DNA.

p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers.

Mutations in the p53 tumor suppressor gene are commonly found in the major human cancers and the mutational spectrum in some cancer types is consistent with the genotoxic effects of the associated environmental risk factors. Thus far there is little information on p53 mutations in cancers of factory workers with a history of carcinogen exposure in the workplace. Occupational exposure to vinyl chloride causes liver angiosarcomas (ASL) and also increases the risk of several other cancers. Loss of p53 function in osteo- and fibrosarcomas can occur by two different mechanisms, p53 mutation and amplification of the MDM2 gene. We examined tumors from five vinyl chloride-exposed patients, four with ASL and one with hepatocellular carcinoma (HCC), for evidence of MDM2 proto-oncogene amplification or p53 mutation in exons 5-8. Amplification of MDM2 was not found, but in two of the angiosarcomas an A:T to T:A missense mutation was detected. p53 sequence analysis of vinyl chloride associated cancers may provide valuable information on the relationship between carcinogen exposure and DNA damage in cancer-related genes.

Nonrandom distribution of O6-methylguanine in H-ras gene sequence from DNA modified with N-methyl-N-nitrosourea.

The distribution of O6-meG in the rat H-ras gene sequence was studied using PCR by transition of O6-meG to adenine during the reaction. In order to study the transition mutations the PCR product was cloned in a replicative form of phage M13mp18 and sequenced. The use of PCR for detection of O6-meG was validated by using oligonucleotides (61 bases) containing one O6-meG residue at a defined site. After treatment of rat liver DNA by N-methyl-N-nitrosourea in vitro, a striking nonrandom sequence distribution of O6-meG was observed. Sixty-eight per cent of O6-methylated Gs were found in the middle G of the sequences GGT and GGA in the H-ras gene whereas no methylation was found in the middle G of the sequences AGG, GGG, TGT, TGC, CGA and CGC. No O6-meG adduct was found in the 12th codon of H-ras (sequence GGA). The frequency of O6-meG formation as a function of two flanking nucleotides on each side of the target guanine was calculated as an approach to understanding more distant sequence effects. It was found that in the DNA sequence studied the formation of O6-meG was highest if the G was flanked by PyPu or PuPu on the 5' side (Py, pyrimidine and Pu, purine) whereas PuPu on the 3' side showed maximal inhibition of O6-meG formation.

Modulation of O6-methylguanine-DNA methyltransferase in rat and hamster liver after treatment with dimethylnitrosamine.

Distinct species differences exist between BDIV rats and Syrian Golden hamsters in the repair of methylated DNA lesions, after single exposures to dimethylnitrosamine (DMN). The promutagenic lesions O6-methylguanine (O6-MeG) and O4-methylthymidine were actively repaired in rat liver; in contrast, in hamster liver the levels of O6-MeG remained relatively stable while O4-methylthymidine levels were reduced. Species differences in the levels of two enzymes involved in the repair of DNA alkylation damage were also noted. An increase in the methylpurine-DNA glycosylase levels was seen in both species following DMN exposure; however, significant species differences in the inactivation and subsequent time course of recovery of the "suicide protein" O6-MeG-DNA methyltransferase were observed. In the rat a rapid recovery of activity began within 24 h of DMN exposure (20 mg/kg) and an approximately 3-fold induction in enzyme levels was observed at 96 h. In hamster liver, in which the constitutive level of expression of this enzyme is similar, no activity was detectable up to 96 h after treatment (25 mg/kg DMN). Only in animals in the lowest treatment group (2.5 mg/kg DMN) was a significant recovery seen, 264 h after treatment. The data presented suggest that the schedule of DMN treatment, in particular the time between doses of the carcinogen and the regeneration of the O6-MeG-DNA methyltransferase, would evoke different carcinogenic responses in hamster and rat liver following chronic exposure to alkylating agents.

Measurement of the removal of O6-methylguanine and O4-methylthymine from oligodeoxynucleotides using an immunoprecipitation technique.

A sensitive and rapid procedure for measurement of alkyltransferase repair activity involving oligodeoxynucleotides followed by immunoprecipitation is described. Dodecadeoxynucleotides containing O6-methylguanine or O4-methylthymine were used as substrates for alkyltransferases and the reaction products of methylated or demethylated substrates were separated by precipitation with highly specific antibodies. This approach for O6-alkylguanine-DNA alkyltransferase measurement is far more rapid than when the reaction products are separated by chromatography. This technique makes the assay applicable to large-scale epidemiological or clinical studies and suggests a similar methodology could be applied for other DNA repair enzymes.

Detection in human cells of alkylated macromolecules attributable to exposure to nitrosamines.

Various methods for detecting DNA alkylation adducts are described briefly, with emphasis on immunoassays using antibodies against O6-methyldeoxyguanosine (O6-medGua), O4-methylthymidine (O4-meThy) and 7-methyldeoxyguanosine (7-medGua). The application of these methods to epidemiological studies is discussed, and results obtained so far on the presence of DNA alkylation adducts in human tissues are presented.