p53 and K-ras mutations in lung cancers from former and never-smoking women.

Somatic p53 mutations are common in lung cancer. Active cigarette smoking is positively correlated with the total frequency of p53 mutations and G:C to T:A transversions on the nontranscribed (DNA coding) strand. Mutational hotspots within the p53 gene, e.g., codon 157, have been identified for tobacco-related lung cancer, whereas these same mutations are found rarely in other cancers. Such data implicate specific p53 mutations as molecular markers of smoking. Because limited data exist concerning the p53 mutation frequency and spectra in ex-smokers and nonsmokers, we have analyzed p53 and K-ras mutations in 126 lung cancers from a population-based case-control study of nonsmoking (n = 117) or ex-smoking (n = 9) women from Missouri with quantitative assessments of exposure to environmental tobacco smoke. Mutations in the p53 gene were found in lung cancers from lifetime nonsmokers (19%) and ex-smokers (67%; odds ratio, 9.08; 95% confidence interval, 2.06-39.98). All deletions were found in tumors from patients who were either ex-smokers or nonsmokers exposed to passive smoking. The G:C to A:T transitions (11 of 28; 39%) were the most frequent p53 mutations found and clustered in tumors from lifetime nonsmokers without passive smoke exposure. The incidence of K-ras codon 12 or 13 mutations was 11% (14 of 115 analyzed) with no difference between long-term ex-smokers and nonsmokers. These and other results indicate that p53 mutations occur more commonly in smokers and ex-smokers than in never-smokers. Such comparisons provide additional evidence of genetic damage caused by tobacco smoke during lung carcinogenesis.

Single-Strand Conformation Polymorphism Analysis of Mutations in Exons 4-8 of the TP53 Gene.

The TP53 tumor suppressor gene coding for a nuclear phosphoprotein involved in cellular stress responses is the most frequently mutated gene in human cancers described so far (1-4). Mutations are found throughout the gene but most frequently within the highly conserved middle region (exons 5-8) that encodes for the DNA-binding central region of the gene critical for the major function of TP53 protein as a transcriptional activator (5). The mutation spectrum of the TP53 gene varies from one tumor type to another with typical hot-spot codons for mutations (2,6). For instance, codons 157, 248, and 273 are frequently mutated in cigarette smoking-associated lung cancer, whereas mutations in codon 175 are rare. This codon, on the other hand, is often mutated in breast and colon cancer. In some cases, typical mutations can be linked with environmental exposures, such as CC→TT double mutation with UV radiation (7) and codon 249 AGG→AGT mutation with aflatoxin B1 and hepatitis B virus (8). These findings, in connection with the fact that one of the main functions of TP53 protein is putatively the protection of the genome (9,10) implicate the mutations of the TP53 gene in environmentally induced carcino-genesis in humans and the possible use of TP53-related markers in molecular epidemiology (11-13).

Environmental tobacco smoke, genetic susceptibility, and risk of lung cancer in never-smoking women.

BACKGROUND: Exposure to environmental tobacco smoke (ETS) is considered to be a major lung cancer risk factor for never smokers. We investigated the hypothesis that never-smoking women who are exposed to ETS and develop lung cancer are a genetically susceptible population. METHODS: Archival tumor tissues were analyzed from 106 never-smoking women enrolled in a case-control study of ETS (and other personal and environmental factors) and lung cancer risk. We analyzed germline polymorphisms in genes that have been associated with cancer susceptibility and whose products activate (cytochrome P450 1A1 [CYP1A1]) and detoxify (glutathione S-transferases M1 [GSTM1] and T1 [GSTT1]) chemical carcinogens found in tobacco smoke. RESULTS: When compared with never smokers who had no ETS exposure and developed lung cancer (n = 55), never smokers with exposure to ETS who developed lung cancer (n = 51) were more likely to be deficient in GSTM1 activity (i.e., were GSTM1 null) because of a genetic polymorphism in the GSTM1 gene (odds ratio = 2.6; 95% confidence interval = 1.1-6.1). A statistically significant rising trend in risk occurred with increasing ETS exposure (two-sided P =. 02), reaching a more than sixfold excess risk in those exposed to 55 pack-years of ETS (ETS pack-year = ETS produced by an active smoker, within a confined space such as a room, who smokes one pack of cigarettes a day for a year). No evidence was found of associations between GSTT1 deficiency or the CYP1A1 valine variant and lung cancer risk due to ETS exposure. CONCLUSIONS: A common genetic polymorphism divides the population of never smokers into two groups of approximately equal size, one (homozygous carriers of the GSTM1 null allele) that has a statistically significant greater risk of lung cancer from ETS than the other (heterozygous or homozygous carriers of the wild-type GSTM1 allele).

Germ-line TP53 mutations in Finnish cancer families exhibiting features of the Li-Fraumeni syndrome and negative for BRCA1 and BRCA2.

Mutations in BRCA1 and BRCA2 account for a large portion of the inherited predisposition to breast and ovarian cancer. It was recently discovered that mutations in these two genes are less common in the Finnish population than expected. Because the genetic background of breast cancer, in particular, is largely obscure, it became necessary to search for mutations in other susceptibility genes. Because seven of our BRCA1 and BRCA2 mutation-negative families fulfilled the criteria of either Li-Fraumeni syndrome (LFS) or Li-Fraumeni-like syndrome (LFL), we decided to screen them for germ-line TP53 mutations in exons 5-8 using a dual-temperature single-strand conformation polymorphism assay (SSCP). Two missense mutations (Asn235Ser and Tyr220Cys) were identified. The clinical significance of these findings was evaluated by comparison to previously reported germ-line TP53 mutation data, and by using the tumor loss of heterozygosity (LOH) analysis. In addition, an immunohistochemical analysis of tumor specimens from mutation-positive individuals was performed. Our results suggest that the observed missense mutations confer susceptibility to cancer, and that germ-line TP53 mutations would therefore explain an additional fraction of hereditary breast cancer in Finland.

p53 status of newly established acute myeloid leukaemia cell lines.

We analysed the status of the p53 gene and protein in eight newly established acute myeloid leukaemia (AML) cell lines representing blast cells of either de novo leukaemia patients in first remission or patients with relapsed and chemotherapy-resistant disease causing their death. There were no mutations in the p53 gene in any of the cell lines as analysed by single-strand conformation polymorphism of amplified exons 5-8. However, the p53 protein was clearly and consistently expressed in all of these cell lines, as shown by immunohistochemistry, Western blotting and flow cytometry. The consistently expressed p53 protein was located in both the nucleus and the cytoplasm of all the cell lines and, as shown by flow cytometry, it was mostly in a conformation typical of the mutated protein. These AML cell lines offer a tool for studying the production and function of the p53 protein and its possible role in cell cycle regulation and chemoresistance as well as in the regulation of apoptosis in AML.

Absence of p53 mutations in benign and pre-malignant male genital lesions with over-expressed p53 protein.

Mutations of the tumor-suppressor gene p53 are common in epithelial tumors. Clonal mutations of p53 have been found in cervical and vulvar carcinomas negative for human papillomavirus (HPV), though at least in cervical cancer HPV infection and p53 mutations are not mutually exclusive. We have previously shown that about 40% of male genital warts and bowenoid papulosis lesions exhibit immunohistochemically detectable aberrant p53 protein, irrespective of the presence of HPV DNA. We studied p53 mutations in exons 4-8 with SSCP and sequencing in 13 male patients with 1 to 3 therapy-resistant genital warts or intra-epithelial neoplasias each and in 4 patients with penile squamous cell carcinoma. Thus, 13 genital warts, 6 bowenoid papulosis, 1 Queyrat's erythroplasia and 1 carcinoma in situ were studied. p53 protein was detected immunohistochemically, and HPV status was analyzed with DNA in situ hybridization and amplification of HPV-specific DNA. There was no correlation between p53 protein expression and HPV status. No mutations in exons 5-8 of the p53 gene were found in any of the lesions, and furthermore, no exon 4 mutations were found in lesions positive in p53 immunohistochemistry. In conclusion, overexpression of p53 does not indicate a p53 mutation in male genital warts, pre-malignant lesions or malignant squamous cell carcinomas. Our study thus suggests that p53 mutations are not important, or at least not early, events in male genital carcinogenesis.

Infrequent p53 mutations in arsenic-related skin lesions.

Oral arsenic exposure increases the risk for a variety of benign and malignant skin lesions, but the molecular mechanism of the carcinogenic effect is poorly understood. Arsenic-related squamous cell carcinomas of the skin can develop either de novo or progress from Bowen's disease lesions. Arsenic-related basal cell carcinomas develop usually in non-sun-exposed areas and are multiple. Because p53 tumor suppressor protein is a protective cellular molecule against environmental carcinogens and mutations in the p53 gene are frequent in nonmelanoma skin cancers, we studied p53 in 23 premalignant or malignant skin lesions from seven patients with a history of arsenic medication. The eighth patient studied (with six lesions) had a long standing exposure to UV radiation. Accumulation of the p53 protein was detected (with a monoclonal DO-7 antibody) in 78% of the lesions from cases with arsenic exposure. Two of the six (30%) arsenic-related premalignant lesions and in addition one UV related carcinoma in situ lesion were clearly and repeatedly positive when p53 exons 5 to 8 were screened by a nonradioactive single-strand conformation polymorphism (SSCP) analysis. Only one of the arsenic-related lesions was confirmed by sequencing to have a mutation (a CC to TT double transition). No indications of mutations were found among the 18 basal cell carcinoma or two squamous cell carcinoma lesions studied. Our results suggest that the frequent accumulation of p53 protein in arsenic-related skin lesions is not due to p53 mutations. which may not be a prerequisite in the development of arsenic-induced skin cancers.

Single-strand conformation polymorphism analysis to detect p53 mutations: characterization and development of controls.

Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified p53 exons 4-8, where mutations frequently occur in human tumors. Easily obtainable and clearly distinguishable positive controls were developed by replacing the regular 5' primers in amplification with primers that include one to three mutated sites. Careful purification of the amplified products by gel electrophoresis appeared to be essential. The efficiency of the method was studied by using previously sequenced samples with p53 mutations and the various positive controls. The use of two temperatures (exon 4: 4 degrees C and 15 degrees C; other exons: 4 degrees C and 20 degrees C) in combination with other optimized conditions resulted in 98% efficiency in mutation detection, which was considered sufficient for routine screening.

Metabolites and DNA-binding of carbamazepine and oxcarbazepine in vitro by rat liver microsomes.

DNA-binding of carbamazepine (CBZ) and oxcarbazepine (OCBZ) catalysed by non-induced, phenobarbital-induced or methylcholanthrene-induced rat liver microsomes in vitro was studied. 14C-CBZ 200 nmol incubated with DNA, liver microsomes and cofactors led to the formation of a significant amount of CBZ-epoxide, which has been suspected as the cause of teratogenesis and other side-effects of CBZ, 1,2 but has not been reactive in any test systems for genotoxicity, including the Ames test.3 No enzyme-dependent DNA-binding of CBZ was found. Using the same conditions, however, OCBZ was bound to DNA. This binding was dependent on the presence of NADPH. 10-hydroxy-10, 11-dihydro-carbamazepine, which is known to be the major metabolite of OCBZ, and an unknown peak were demonstrated by HPLC. These results are the first indication of a higher level of covalent DNA binding of OCBZ than of CBZ. The nature of the unknown metabolite and the pathway leading to covalent binding remain to be studied.

p53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines.

p53 inhibits cell cycle progression and DNA damaging cytostatics induce p53 protein expression, indicating that p53 responds to DNA damage. We have measured benzo[a]pyrene (BP)-induced DNA damage in association with p53 expression. The most relevant DNA adducts for carcinogenesis, benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts, were measured by synchronous fluorescence spectrophotometry and p53 immunohistochemistry using polyclonal antibody CM1, which detects both wild-type and mutated forms of p53. Activation of BP in A-549 lung carcinoma and MCF-7 breast adenocarcinoma cell lines containing wild-type p53 was followed by an increase in p53 protein expression. alpha-Naphthoflavone, an inhibitor of cytochrome P450 (CYP)1A1, decreased both the formation of diolepoxide metabolites and the p53 response. The cell lines not able to activate BP, A-427 and SK-LU-1 (both human lung carcinomas), SK-MES-1 (human lung squamous carcinoma) and human fibroblasts, did not show any increase in p53 immunohistochemistry. The OVCAR-3 ovarian adenocarcinoma cell line, containing a mutation in exon 7 of p53, and the SK-LU-1 cell line expressed very high levels of p53 protein before BP treatment and no increase in p53 immunohistochemistry was seen. These findings indicate that p53 protein is part of the response of the cells to BP-induced DNA damage.