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[Cancer Research 61, 4350-4356, June 1, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

p53 and K-ras Mutations in Lung Cancers from Former and Never-Smoking Women1

Kirsi H. Vähäkangas2,,3, William P. Bennett2, Katariina Castrén, Judith A. Welsh, Mohammed A. Khan, Brunhilde Blömeke4, Michael C. R. Alavanja and Curtis C. Harris5

Department of Pharmacology and Toxicology, University of Oulu, P. O. Box 5000, FIN-90014 University of Oulu, Finland [K. H. V., K. C.]; Division of Molecular Medicine, City of Hope/Beckman Research Institute, Fox Plaza South, Duarte, California 91010-3000 [W. P. B.]; Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892 [J. A. W., M. A. K., B. B., C. C. H.]; and Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. C. R. A.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the United States, lung cancer accounts for ~28% of the cancer deaths, and smoking is acknowledged as the prominent cause of this disease (1, 2, 3) . Lung cancer incidence rates for women in the United States continue to climb, whereas for men, a downward trend began several years ago (3) . Although the increased risk of lung cancer is less after smoking cessation, it never regains the level of life-long never-smokers, indicating permanent damage in lung tissue from smoking (4) . Environmental cigarette smoke from household and workplace exposures also increases the risk of lung cancer in never-smokers (1 , 5, 6, 7, 8, 9) . The number of epidemiological studies published on the effect of passive smoking on the risk of lung cancer has increased from the three by 1982 to 37 by 1997 (reviewed in Ref. 10 ), and the meta-analysis of these studies indicates an excess risk with a dose-response relationship from both the number of cigarettes and the length of exposure (11) . These epidemiological data are consistent with experimental studies on environmental cigarette smoke (12) and with studies indicating that sidestream cigarette smoke contains certain carcinogens at higher concentrations than those formed in the mainstream smoke (1) .

Lung carcinogenesis involves a multitude of genetic and epigenetic changes (13, 14, 15, 16) including loss of heterozygosity at 3p, 5q, 8p, 9p (p16/CDKN2), 9q, 11p, 11q, 13q (Rb),6 17p (p53), and 17q, known and putative sites of tumor suppressor genes (17 , 18) . The major genetic alterations in lung cancer detected thus far include p53 gene mutations; inactivation of the Rb pathway including p16INK4 and Rb mutations; K-ras mutations and the disruption of fragile histidine triad sequences (recently reviewed in Ref. 15 ). The p53 gene on chromosome 17p13.1 codes for the nuclear phosphoprotein p53, a transcription factor involved in the regulation of the cell cycle and apoptosis, and is mutated frequently in human cancers, e.g., >50% of lung cancers carry such a mutation (reviewed in Refs. 19, 20, 21 ). p53 mutations also have been found in premalignant lesions in lung tissue and indicate an early loss of this tumor suppressor’s function in lung carcinogenesis (22, 23, 24) .

Active cigarette smoking is positively correlated with the total frequency of p53 mutations and G:C to T:A transversions on the nontranscribed strand of the p53 gene (for recent reviews on p53 mutation spectra, see Refs. 25, 26, 27, 28, 29 ). There also are mutational hotspots in the p53 gene in lung cancer (e.g., codon 157), but these same hotspots are rarely found in other types of cancers, thus suggesting that different mutagens cause different mutations. Perhaps the most convincing evidence linking tobacco carcinogens to direct genetic damage of p53 coding sequences is the accumulation of BPDE adducts at mutational hotspots within codons 157, 248, and 273 (30) and the induction of p53 mutations, including codon 248, in human cells exposed to benzo(a)pyrene (31) . BPDE adducts at these codons also are repaired poorly (32) . Among the major hotspots within the p53 gene, the methylated CpG sites constitute the majority (19 , 21 , 27 , 28) . Recently, cytosine methylation at CpG sites was reported to enhance the binding of carcinogens such as benzo(a)pyrene to the guanine sites opposite of the 5-methyl-cytosines in the p53 gene (33 , 34) .

Although p53 mutation data are available from >1000 lung cancers, only a small portion of these are p53 mutations in defined nonsmokers (29) . Therefore, we have analyzed p53 mutations in 117 lung cancers from a population-based case-control study of nonsmoking women from Missouri and nine ex-smokers from the same population (5) . p53 mutations were compared with K-ras mutations. IHC analysis of the p53 protein was carried out as an additional screening method with SSCP, because IHC positivity has been regarded as an indicator of a p53 mutation. However, it is known that mutation is only one mechanism that increases p53 protein in cells (35) . Frequencies of specific mutations in the p53 gene were compared with the ones in the revised IARC database (29) , which is the largest existing p53 mutation database. Results from epidemiological investigations on the same population have been reported for passive-smoking exposure (5) , home radon concentrations (36) , dietary practices (37) , occupational risk factors (38) , and nonneoplastic disease (39) . In a separate report (25) , we have presented evidence that homozygous deletion of GSTM1 coding sequences confers a significant risk of lung cancer to lifetime nonsmokers exposed to ETS.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Series and Study Population.
The study population included 131 Caucasian women from Missouri who were 41–84 years of age at the time of lung cancer diagnosis (Table 1Citation ; five cases were excluded from the analysis; see "Reconsideration of Nonamplified Exons"). The study was restricted to Caucasian women because of the low numbers of other ethnic groups and male nonsmokers. Information on active and passive smoking, dietary preferences, inflammatory lung disease, and other risk factors for lung cancer were collected by trained interviewers using a structured questionnaire as described previously (6 , 36 , 37) . Nonsmokers were defined as persons who had neither smoked >100 cigarettes nor used any tobacco products >6 months during their lifetimes (6) . Eleven ex-smokers stopped smoking at least 15 years before the study interview, and their smoking histories ranged from 1 to 105 pack-years. Quantitative exposure to ETS, measured in ETS pack-years, was determined by telephone interviews that documented the source (e.g., parent or spouse), intensity, and duration of exposure during childhood and adulthood (5) . One ETS pack-year is the exposure, within a confined space such as a room, to ETS produced by an active smoker consuming one pack of 20 cigarettes each day for a year. Passive smoking data were available in 96% (126 of 131) of cases, and for analytic purposes, the population was divided into four groups as follows: 0 pack-years, <21 pack-years, 21–52 pack-years, and >52 pack-years. Measurements of home radon gas concentrations were performed by placing {alpha}-track detectors in each dwelling that was occupied for at least 1 year by the study subject during the preceding 30 years (36) . Home radon measurements were completed in 92% (120 of 131) of these cases, and 83% had <2.5 pCi/l, which is typical for American homes and well below the EPA action level of 4.0 pCi/l. For statistical analyses, the population was divided into quartiles defined by the following breakpoints: 0–0.9, 0.9–1.5, 1.5–2.2, and >2.2 pCi/liter.


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  		Table 1 Association of p53 mutation status with patient characteristics

 
Mutation Analysis of the p53 Gene.
Complete coding sequences and splice junctions for exons 5–8 were screened for mutations by SSCP analysis (40, 41, 42, 43) . This newly developed, nonradioactive assay includes analysis at two temperatures, which ensures a high efficiency for mutation detection. DNA was extracted from microdissected, paraffin-embedded tumor sections by a treatment with proteinase K for 3–5 days at 37°C and phenol extraction-ethanol precipitation method. For amplification of p53 exons 5–8, two sets of intronic primers (44) , the second set internal to the first one for each exon, were used in two consecutive amplification reactions. One hundred-µl reactions contained either Dynazyme (Finnzymes, Espoo, Finland) with the recommended buffer or AmpliTaq (Perkin-Elmer, Branchburg, NJ) with a 10x buffer containing 500 mM Tris (pH 9) and 30 mM MgCl2 with 20–30 pmol of each primer and 300 µM of dNTPs (Pharmacia Biotech). Two control reactions without a template were included in each set of amplifications to rule out contamination. Amplified products were gel-purified on a 4% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME). Purified samples were analyzed by SSCP using a temperature-controlled electrophoresis system (PhastSystem; Pharmacia Biotech, Piscataway, NJ), PhastGel Homogenous 20% polyacrylamide gels, and native buffer strips. Gels were stained with PhastGel DNA Silver Staining kit (Pharmacia Biotech). Amplified DNA using artificially mutated nested primers for positive controls and confirmed WT lymphocyte DNA for negative controls were included in each SSCP run.

As the second step, all of the repeat and similar positive cases by PCR-SSCP, as well as those showing high positivity by p53 IHC, were sequenced manually. Direct radioactive dideoxy-sequencing on both the coding and noncoding strands, using the same amplified samples as for SSCP, was done using a Sequenase kit from Amersham. To maximize the labeling, 35S-labeled dNTP was selected according to the first nucleotide after the primer in each case. After denaturation at 100°C for 3 min, the sequencing reactions were carried out at 42°C. In p53 mutation analysis, at least two independently amplified DNA samples with identical results were required for any sample to be labeled "mutated."

Analysis of p53 Mutation Database.
Lung cancer mutations were graciously provided by Drs. P. Hainaut and G. P. Pfeifer from the recently revised IARC database (29) . In this version of the database, the lung cancer mutation entries have been edited to remove duplicate entries and 107 mutations from an anomalous report. In addition, the demographic information and smoking histories have been verified and corrected by manual review of the original publications. Before compiling data shown in Figs. 1Citation and 2Citation , we excluded mutations from lung cancers associated with occupational exposures (e.g., chromate, asbestos, silicates, and mustard gas) or ionizing radiation (e.g., uranium mining, cancer therapy, and A-bomb survivors).



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  		Fig. 1. p53 mutation spectra in lung cancers from smokers, ex-smokers, and nonsmokers. Because of numerical rounding by Microsoft Excel, percentages in figures may differ from those calculated in the text. A, lifetime nonsmokers in this study. B, female nonsmokers from the revised IARC database (29) . C, male and female nonsmokers from the revised IARC database: 69 females, 22 males, and six of unknown gender (29) . D, ex-smokers; combined data from this study with those in the revised IARC p53 mutation database (29) : 12 females, nine males, and five of unknown gender. E, smokers from the revised IARC p53 mutation database (29) : 64 females, 186 males, and 79 of unknown gender. F, heavy smokers from the revised IARC database (29) : three males and 17 of unknown gender.

 


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  		Fig. 2. Distribution of p53 gene mutations in lung cancers from female nonsmokers (n = 22) in this study compared with the reported data from female smokers (n = 64) in the revised IARC-based p53 mutation database (29) .

 
Reconsideration of Nonamplified Exons.
There were some samples where, despite repeated amplifications from several tissue sections, one or more exons of p53 did not amplify. In each case, some exons of p53 amplified, and we also were able to amplify K-ras in these cases, which indicates DNA of sufficient quality for PCR amplifications. Thus, among the 131 cases, 13 miss the mutational data for one exon of p53, two cases for two exons, and three cases for three exons. One case with one "missing exon" contained a mutation in another exon. It is possible that in these cases there was a deletion of part of the p53 gene, a mutation, or a deletion at the site of the PCR primer. Calculations of p53 mutation frequencies are based on 117 cases of lifetime nonsmokers (104 cases with four exons and 13 cases with three exons of p53 analyzed) and nine cases of ex-smokers (four exons were analyzed in all cases).

p53 Immunohistochemistry.
As described previously (45) , DO7 monoclonal antibody raised against human WT p53, which recognizes both WT and mutated p53 in IHC, was used as the primary antibody in immunohistochemistry. Each case was stained at least twice, and the coded slides were read by three pathologists for a consensus of the extent of positivity.

Mutation Analysis for K-ras.
The sequence area around codons 12 and 13 of K-ras was amplified. Amplified strands were gel-purified and sequenced using direct dideoxy-sequencing as described above. PCR amplification was performed in a two-step amplification using external and nested primer sets: external forward (5'-GTACTGGTGGAGTATTTGAT-3') and external reverse (5'-TGCACCTAGGACTCATGAAAATGGTCAGAG-3'); and nested forward (5'-ACATGTTCTAATATAGTCAC-3') and nested reverse (5'-CTATTGTTGGATCATATTCG-3').

The initial PCR amplification was done in a 100-µl reaction mixture, with 100–300 ng of genomic DNA as template. The nested PCR was done using a 300-µl reaction mixture. PCR products were generated by using primers at a final concentration of 20 pmol/100 µl. Amplification was performed with Taq polymerase (2.5 units/100-µl reaction mixture; Perkin-Elmer, Branchburg, NJ) in a Perkin-Elmer DNA thermal cycler using 35 PCR cycles preceded by a "hot start" of 95°C for 5 min. Each PCR cycle consisted of 1 min at 94°C, 1 min at 50°C, and 30 s at 72°C. The PCR buffer consisted of final concentrations of 50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3 at room temperature), 15 mM magnesium chloride, and 7.5 mM ammonium sulfate; each dNTP final concentration was 200 µmol/liter (800 µmol/l for all four dNTPs). The final PCR amplified product was 182-bp fragments encompassing nucleotides 42–204 of exon 1 of the K-ras gene. The amplified PCR products were electrophoresed in agarose (NuSieve & SeaKem, FMC Corp., Rockland, ME) containing ethidium bromide, run at 150 V for 45 min, and visualized under UV.

Direct sequencing was performed using 5 µl of the amplified product with 35S-labeled nucleotide. The sequencing method was similar to the one described for p53, except nested primers for K-ras were used. Sequences were confirmed by sequencing both strands of the DNA and a positive result by new amplification.

Statistical Analysis.
Mutational spectra were compared using the Monte Carlo method with the Adams and Skopek algorithm as described by Piegorsch and Bailer (40) using a computer program created by Cariello et al. (46) .

Maximum likelihood procedures were used in computing odds ratios and 95% confidence intervals. Multivariate logistic regression models were used to adjust for potentially confounding variables: age, GSTM1 status, vegetable consumption, and domestic radon exposure.

Wald confidence intervals were computed on the bases of the estimated parameter ß, and its SE (47) :


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To our knowledge, this is the largest series of p53 mutations in lung cancers from never-smoking women enrolled in a population-based study of passive smoking. To better understand our results, we have compared them with the revised IARC-hosted database of p53 mutations in lung cancer (29) , which has information of smoking status in 451 cases of the 1346 lung cancers with p53 mutations. The database contains 64 mutations in lung cancers from female smokers and 97 mutations in lung cancers from nonsmokers, of which 69 are female nonsmokers.

p53 Mutations in Lifetime Nonsmokers.
Twenty-two of the mutations were in lung cancers from 117 lifetime nonsmokers (19%), where three exons (13 cases) or all four exons (104 cases) could be amplified for analysis. The percentage of G:C to A:T mutations of all found mutations was 41% (9 of 22) in lifetime nonsmokers (Fig. 1)Citation . Six of 22 (27%) of the mutations in lifetime nonsmokers were G:C to A:T at the CpG sequences. Such mutations are not as common in smokers, especially male smokers (29) . Six of 22 (27%) of the p53 mutations in the lifetime nonsmokers were at the hotspot codons reported in lung cancer, and only one of the hotspot codon mutations was a G->T transversion (Table 2Citation ; Fig. 2Citation ). Two mutations were found at codon 241 and again at codon 242. Codon 241 rarely is mutated in lung cancer (seven reported mutations in the revised IARC database; Ref. 29 ).


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  		Table 2 p53 mutations in never-smoker lung cancers

 
p53 Mutations in Ex-Smokers.
Six p53 mutations were detected in 11 ex-smokers (55%). If the calculation is based on cases with all four exons amplifiable (in one case only one exon, and in one case two exons were amplifiable), the percentage is even higher (6 of 9; 67%). Three of six (50%) p53 mutations in the ex-smokers were at the hotspot codons in the p53 gene reported in the literature, and one of these was a G:C to T:A transversion (Table 3)Citation . The percentage of hotspot mutations in ex-smokers was higher than the percentage in lung cancers from lifetime nonsmokers in this study (Tables 2Citation and 3Citation ; Fig. 2Citation ). The proportion of cases with a p53 mutation was much higher in lung cancers from ex-smokers than in lung cancers from lifetime nonsmokers (highly statistically significant difference with age adjusted odds ratio of 9.08 and 95% confidence interval of 2.06–39.98). Adjustment for vegetable consumption, GSTM1 status, or radon exposure did not alter the risk estimate. All four deletions were found in cancers from ex-smokers or those nonsmokers exposed to ETS.


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  		Table 3 p53 mutations in ex-smoker lung cancers

 
p53 Immunopositivity and Histology.
Immunopositivity of the p53 protein was detected in 76 of the 131 samples (58%). A statistically positive correlation between IHC positivity and p53 mutations was not observed. Most of the lung tumors in our study (73%) were adenocarcinomas. Adenocarcinoma is the most common histological subtype in nonsmoking women (48) . The percentage of p53 mutations in adenocarcinomas in our study is 25% (23 of 92).

p53 Mutations in Relation to Radon Exposure.
Twenty-four of the 28 p53 mutations were found in lung tumors from the 120 cases with known indoor radon exposure measurements. No statistically significant differences were found among the different radon quartiles (quartile 1, 6 of 30, 20%; quartile 2, 9 of 31, 29%; quartile 3, 4 of 28, 14%; quartile 4, 5 of 28, 18%).

K-ras Mutations.
None of the cancers carried more than one p53 mutation. However, in five cases, the tumor sample contained a K-ras mutation in addition to the p53 mutation (Table 4)Citation . The incidence of K-ras codon 12 or 13 mutations among these cases was 11% (14 mutations in 126 successfully analyzed cases) with no difference between long-term ex-smokers (1 of 9; 11%) and lifetime nonsmokers (13 of 117; 11%). Thirteen of the mutations were found at codon 12, and only 1 K-ras mutation was found at codon 13 in an ex-smoker (Table 4)Citation . Six codon 12 mutations occurred in nonsmokers who had no passive smoking exposure. Only one (7%) of the K-ras mutations was a G:C to T:A transversion, which is the most frequent K-ras mutation reported in smokers (57%; Ref. 49 ), but it was found in a tumor sample from a person exposed to passive smoking. The most frequent type of base change was a G:C to A:T transition (7 of 14 mutations).


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  		Table 4 K-ras mutations in nonsmoker lung cancers

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study is consistent with published data indicating a different p53 mutation spectrum in lung cancers from nonsmokers when compared with lung cancers from smokers (16 , 21 , 26 27, 28 , 50) . Differences can be identified in the frequency, type, and distribution of p53 mutations. A significant finding in this study was the lower frequency of p53 mutations in lifetime nonsmokers (22 of 117; 19%) when compared with the frequency of ex-smokers (6 of 9; 67%; P = 0.016). The p53 gene mutation frequency that we found in nonsmokers (19%) was similar to previous reports 7 of 35 (20%; Ref. 51 ); 6 of 23 (26%; Ref. 52 ); 18 of 69 (26%; Ref. 53 ); and 9 of 91 (10%; Ref. 54 ). The frequency of p53 mutations in ex-smokers in this study (6 of 9; 67%) is similar to that in smokers reported by Greenblatt et al. (21) and Hernandez-Boussard and Hainaut (26) but higher than the 29 or 38% found in ex-smokers by Husgafvel-Pursiainen et al. (54) . More p53 mutations were found at known lung cancer hotspots in ex-smokers than in lifetime nonsmokers in this study. Because the ex-smokers had stopped smoking >15 years prior to the surgical removal of the lung cancer, our findings indicate the persistence of molecular damage from smoking. This conclusion also is supported by the findings of loss of heterozygosity and microsatellite alterations, which persist in bronchial epithelium years after smoking cessation (12 , 55) .

In accordance with the revised IARC database information (29) , the frequency of G:C to T:A transversions in the p53 gene of female lifetime nonsmokers reported here is less [4 of 22 (18% of all mutations); 4 of 19 (21%) missense mutations] than in lung cancers from smokers (26% in all smokers and 29% in female smokers; Ref. 29 ; Fig. 1Citation ). Although these mutations have been reported to be more frequent in lung cancers from female smokers (56 , 57) , such a gender difference was not found in the revised IARC-based p53 mutation database (Ref. 29 ; Fig. 1Citation ). The number of studies on p53 mutations in lung cancer from nonsmokers is still small when compared with the number of studies from smokers, but the published studies agree with each other on the key findings. Our current data, along with other studies of nonsmoker lung cancers (51, 52, 53, 54 , 58, 59, 60) , are in agreement with the hypothesis that the proportion of G:C to T:A transversions is significantly lower in nonsmokers, in which the majority are G:C to A:T transitions. Huang et al. (59) found only one G:C to T:A transversion on the coding strand among the 16 p53 mutations from nonsmokers, whereas all of the G:C to T:A transversions occurred on the coding strand of p53 in Takagi et al. (53) , as well as among our cases. The p53 mutations among nonsmokers from the revised IARC database (29) include a slightly lower percentage of deletion/insertions than the p53 mutations from smokers. In this study, p53 deletions were totally absent in the 11 mutated tumor samples from never-smokers who were without passive smoking exposure. Unfortunately, only a few cases archived in the revised IARC p53 database (29) have information about passive smoking.

The distribution of p53 gene mutations in our study is different from lung cancers in smokers (Refs. 26 and 29 ; Fig. 2Citation ). Although the total percentage of hotspot mutations in our study (32%) was comparable with that reported for lung cancer in general (33%), several hotspots (codons 157, 158, and 249) remained unchanged in these tumors. Although the total percentage of p53 mutations at CpG sites did not differ from previous lung cancer reports (26 , 27) , the percentage in ex-smokers in our study was clearly higher than that in lifetime nonsmokers ((Tables 2Citation and 3Citation ). Our findings fit a current model of lung carcinogenesis whereby certain chemical carcinogens, including polycyclic aromatic hydrocarbons, such as BPDE, preferentially bind to specific DNA sequence motifs to generate discrete mutational hotspots in tobacco-associated lung cancers (30) . More recent investigations have demonstrated that the hotspots are typically CpG dinucleotides containing 5-methyl cytosine (33 , 34 , 61) .

The p53 mutation spectra contain some conspicuous differences between nonsmokers and smokers, thus implicating tobacco carcinogens as etiological factors in lung cancer. On the basis of earlier human studies and this one, a p53 mutation, especially a G:C to T:A transversion, indicates alteration by tobacco carcinogens (29) . In experimental models, this mutation is preferentially caused by BPDE, regardless of the gene studied (35) . On the other hand, although higher doses of BPDE induce mutations, especially in the G:C bp, the proportion of A:T bp mutations increases when the concentration of BPDE decreases (Ref. 62 and references therein). G:C to A:T at CpG sites are typically spontaneous mutations in animal models, whereas N-nitrosamines induce G:C to A:T transitions at non-CpG sites in animals (63, 64, 65, 66) . Nitrosamines from cigarette smoke or from other environmental sources probably contribute to human lung cancer, because G:C to A:T transitions at non-CpG sites are fairly common in lung cancers from both smokers and nonsmokers (Fig. 1)Citation .

A p53 gene mutation is one of the most frequent genetic events in lung cancer with different prevalences, depending on the tumor histology. Adenocarcinomas are especially common in nonsmokers (48) and also formed a majority among our cases (73%). Our results agree with the reported percentages of p53 mutations in lung adenocarcinomas, which have been reported to vary from 20% (51 , 53) to ~33% (21) . Marchetti et al. (51) found no difference in p53 mutation frequency in adenocarcinomas between smokers and nonsmokers.

One hundred and nineteen cases had been analyzed for the GSTM1 polymorphism in our former study of this population (25) , of which, 69 cases were null genotype. Seventeen % of these patients had a p53 mutation in their tumor sample, whereas 28% of those with GSTM1 present had a p53 mutation (Tables 2Citation and 3Citation ). Thus, the GSTM1 null genotype was associated more with WT p53 cases than with mutant p53, and this study and others (67, 68, 69, 70) do not support previous reports indicating the absence of the GSTM1 gene as a susceptibility factor for p53 mutations (71 , 72) .

In smoking-associated adenocarcinoma of the lung, G->T transversions represent the majority (up to 80%) of the K-ras mutations (49 , 73 , 74) . In our study, a K-ras mutation was found in 11% of the lung cancers, with no difference between lifetime nonsmokers and ex-smokers. Only one of the mutations was a G:C to T:A transversion. Whereas the percentage of p53 mutations was lower in nonsmokers than ex-smokers, no difference in the rate of K-ras mutations was suggested in our tumor series. In the literature, a clear association of K-ras mutations with heavy lifetime smoking has been reported (49 , 52) , and it may be that an induction of a p53 mutation requires lower cigarette smoke exposure when compared with a smoke-induced K-ras mutation. On the other hand, Westra et al. (75) found no difference in the frequency of K-ras mutations in lung adenocarcinomas from persistent smokers (30%) versus former smokers (32%).

In conclusion, p53 gene mutations are less frequent and the mutation spectra are different in lung cancers of lifetime nonsmokers when compared with smokers. Lung cancers from ex-smokers contain a p53 mutation spectrum similar to that in smokers, indicating that tobacco carcinogens cause permanent, identifiable damage in lung tissue that can be found decades after smoking cessation. Further studies on molecular markers and the possible long-term effects of past and passive smoking are warranted.


   ACKNOWLEDGMENTS
 
We thank Drs. Pierre Hainaut and Gerd P. Pfeifer for being able to use the revised IARC p53 lung cancer mutation database (29) . We also thank Dr. Zhang Ting-Qiu, Shanghai Medical University, Shanghai, PRC (China) for microdissection of samples, Dr. Eric Berger for computer support, Tuulikki Kärnä (Department of Pharmacology and Toxicology, University of Oulu), and Michael Bartel (Laboratory of Human Carcinogenesis, National Cancer Institute, NIH) for taking part in the p53 mutation analysis, and Drs. Louise Burke and Doug Flieder (Laboratory of Human Carcinogenesis, National Cancer Institute, NIH) for evaluations of the p53 IHC. We also thank Dorothea Dudek for editorial assistance. Finally, we thank Dr. Ross C. Brownson and his collaborators for their foresight.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 K. H. V. was supported with grants from the Finnish Cancer Societies, the Cancer Society in Northern Finland, and the University of Oulu. K. C. was supported with travel grants from the Urho Känkänen and Ida Montin Foundations in Finland and the Finnish Cancer Societies and held a position at the Medical Graduate School of the University of Oulu during these studies. The initial epidemiology study was done in collaboration with Dr. Ross C. Brownson. Back

2 These authors contributed equally to this work. Back

3 Present address: Department of Pharmacology and Toxicology, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland. Back

4 Present address: Department of Dermatology, RWTH-Aachen, 52074 Germany. Back

5 To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Building 37, Room 2C05, MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-2048; Fax: (301) 496-0497; E-mail: Curtis_Harris{at}nih.gov Back

6 The abbreviations used are: Rb, retinoblastoma; IHC, immunohistochemistry; GST, glutathione S-transferase; ETS, environmental tobacco smoke; BPDE, benzo(a)pyrene diolepoxide; SSCP, single-strand conformation polymorphism; WT, wild-type; dNTP, deoxynucleotide triphosphate. Back

Received 2/ 4/00. Accepted 4/ 3/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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