Characteristics of mutations in the p53 gene in oral squamous cell carcinoma associated with betel quid chewing and cigarette smoking in Taiwanese

Ling-Ling Hsieh1,6, Pei-Feng Wang1, I.-How Chen2,6, Chun-Ta Liao2,6, Hung-Ming Wang3,6, Ming-Chi Chen1,6, Joseph Tung-Chieh Chang4,6 and Ann-Joy Cheng5,6

1 Department of Public Health, Chang Gung University, Tao-Yuan, Taiwan,
2 Department of Otolaryngology Oncology, Head and Neck Surgery, Tao-Yuan, Taiwan,
3 Division of Hematology/Oncology Department of Internal Medicine, Tao-Yuan, Taiwan,
4 Department of Radiation Oncology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan,
5 Department of Medical Technology, Chang Gung University, Tao-Yuan, Taiwan and
6 Taipei CGMH Head and Neck Oncology Group, Tao-Yuan, Taiwan
To whom correspondence should be addressedEmail: llhsiel{at}mail.cgu.edu.tw


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
p53 mutations are etiologically associated with the development of oral squamous cell carcinomas (OSCCs) or are associated with exposure to specific carcinogens. In this study, we used PCR–single strand conformation polymorphism and DNA sequencing to analyze the conserved regions of the p53 gene (exons 5–9) in OSCC tumor specimens from 187 patients with varied histories of betel quid, tobacco and alcohol use. Ninety-one of the 187 OSCCs (48.66%) showed p53 gene mutations at exons 5–9. The incidence of p53 mutations was not associated with age, sex, TNM stage, status of cigarette smoking or betel quid chewing. However, alcohol drinkers exhibited a significantly higher incidence (57/101, 56.44%) of p53 mutations than non-users (39.53%, 34/86) (P = 0.02). The effect of alcohol on the incidence of p53 mutations was still statistically significant (RR = 2.24; 95% CI, 1.21–4.15) after adjustment for cigarette smoking and betel quid (BQ) chewing. G:C to A:T transitions were the predominant mutations observed and associated with BQ and tobacco use. Alcohol drinking could enhance these transitions. After adjustment for cigarette smoking and BQ chewing, alcohol drinking still showed an independent effect on G:C to A:T transitions (RR = 2.41; 95% CI, 1.01–5.74). These findings strongly suggest an important contributive role of tobacco carcinogens to p53 mutation in this series of Taiwanese OSCCs and alcohol might enhance these mutagenic effects. As safrole–DNA adducts have been detected in 77% (23/30) of the OSCC tissues from Taiwanese oral cancer patients with a BQ chewing history, we cannot rule out the possibility that safrole or other carcinogens present in the BQ may cause a similar pattern of mutagenesis. Determination of the role of safrole and other carcinogens present in BQ on the pattern of p53 gene mutation in OSCC will require further study.

Abbreviations: B[a]P, benzo[a]pyrene; ; BQ, betel quid; ; OSCC, oral squamous cell carcinomas; ; SSCP, single-stranded conformation polymorphism.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oral cancer is the sixth most common cancer in the world (1). The etiology of oral cancer, specifically cancer of the tongue (ICD9 141) and mouth (ICD9 143–145), is well established and predominantly involves the use of tobacco and alcohol. The prevalence of the disease in different parts of the world reflects different forms and extents of exposure to these etiological agents. In Europe, USA, Australia, China and Japan, cigarette, cigar and pipe smoking are the main forms of tobacco use and the effect of tobacco is known, not only to be dose and time dependent but, also, to act synergistically with the intake of alcohol to multiply disease risk (2). In contrast, in India, Sri Lanka, Papua New Guinea and South East Asia, tobacco chewing is prevalent, usually in the form of a betel quid (BQ) that consists of the leaf of the betel vine (Piper betle) wrapped around areca nut, lime and tobacco; any combination of these constituents may be used. Spices such as cardamom, cloves, aniseed and turmeric, or extracts of Acacia catechu or Acacia suma, may also be incorporated for additional flavor (3). Other forms of tobacco use which are also potent risk factors for oral cancer include the use of nass (an aqueous or oily mixture of tobacco, ash and lime), smoking of bedi (cheap cigarettes in which tobacco is rolled in a temburni leaf) and reverse smoking in which the lighted end of a cigarette or cheroot is held within the mouth (4). In addition, factors such as dietary deficiencies, in particular vitamins A and C, iron and certain trace elements (5), are thought to be associated with oral cancer.

In Taiwan, the incidence of oral cancer has surpassed that of nasopharyngeal cancer and become the fifth most common cancer since 1991 in men. In 1997, the incidence of oral cancer further surpassed stomach cancer and became the fourth most common cancer in men. Ko et al. (6) found that the incidence of oral cancer in Taiwan was 123-fold higher in patients who smoked, drank alcohol and chewed BQ than in abstainers. The odds ratios (ORs) of patients who indulged in at least two of the three habits were significantly elevated as compared with the ORs of patients with a single habit. Furthermore, a statistically significant association between oral cancer and BQ chewing alone was also found. In Taiwan, the number of BQ chewers was estimated at 2.0 million among the 20 million inhabitants in 1992, and the number is still climbing (7). BQ used in Taiwan is different from that used in other countries. BQ used in Taiwan contains areca (betel) nut, slaked lime, catechu, Piper betle inflorescence or Piper betle leaves. This combination is different from that consumed in other countries in three respects. First, tobacco is not included in the chewing of BQ. Second, fresh Piper betle inflorescence is added to BQ for its aromatic flavor. This is not used elsewhere except in Guam and Papua New Guinea (8). Third, fresh and tender areca nut with husk is used in BQ chewing in Taiwan as compared with the ripe and husk removed areca nut used in other countries. Consequently, the mechanisms behind the BQ-related oral cancer in Taiwan may differ from other countries.

Previous studies have shown that certain carcinogens may induce a `fingerprint'-like pattern of mutations at the p53 gene, both in terms of mutation type and codon specificity (9). The most striking example is the p53 mutational spectrum found in hepatocellular carcinoma from either Qidong, People's Republic of China (10,11) or Southern Africa (12,13). A G:C to T:A transversion at the third base position of codon 249 of the p53 gene is strongly associated with dietary aflatoxin intake and hepatitis B virus infection. This type of mutation is consistent with mutations caused in vitro by aflatoxin B1 (14,15). Hence, the mutation spectrum associated with a human cancer can provide clues as to the nature of the incriminating carcinogens and the mutagenic mechanisms responsible for the genetic lesions that drive human carcinogenesis.

The role of p53 mutation in the etiology of oral cancer has not been rigorously studied in Taiwan. Previous studies have shown a lower incidence of mutation associated with BQ chewing in Taiwan (16,17). However, these studies have been based on an analysis of relatively few cases of oral squamous cell carcinomas (OSCCs). In addition, correlations between the observed pattern or incidence of p53 mutations in OSCC with associated risk factors or carcinogen exposure were not explored. In this study, we examined tumor tissues from 187 patients with primary OSCCs for mutations in the conserved regions of the p53 gene and analyzed the pattern and incidence of mutations for correlations with patient tobacco, alcohol and BQ use.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients, tissue specimens and clinical diagnosis
Two hundred and seven oral cancer patients treated at Chang Gung Memorial Hospital, Lin-Kuo during the period from March 1999 through February 2000 were recruited for participation in the study. All patients gave informed consent for participation and were interviewed uniformly before surgery by a well-trained interviewer. The questionnaire used in the interview sought detailed information on current and past cigarette smoking, alcohol drinking and BQ chewing habits, occupational history, family disease history, dietary history and general demographic data.

For each case, a pair of tumor and normal adjacent non-tumor tissue samples were surgically dissected into small pieces, frozen immediately in liquid nitrogen and stored at –80°C. Surgically removed samples were sent to the Department of Pathology for examination and scored according to the recommendations for the reporting of specimens containing oral cavity and oropharynx neoplasms by the Association of Directors of Anatomic and Surgical Pathology (ADASP) (18). Histology diagnosis was categorized as squamous cell carcinoma, verrucous carcinoma, cylindrical cell carcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma or adenocarcinoma. Nine tumor tissues were not the primary cancer lesions and were excluded from the study. Patients with a diagnosis of non-squamous cell carcinoma (n = 11) were also excluded. Thus, a total of 187 patients with primary OSCC (176 males and 11 females) were included in the present study.

Tobacco, BQ and alcohol use. Study participants were asked if they had ever smoked cigarette, chewed BQ and had alcohol on a regular basis (at least once a week). Those who responded `yes' to these questions were classified as tobacco, BQ and alcohol users.

Experimental design. Single-stranded conformation polymorphism (SSCP) analysis was used to analyze all tumor samples for mutations within exons 5–9 of the p53 gene, which are the regions most frequently affected by mutations in human tumors. Cases displaying an altered electrophoretic mobility were re-amplified in another separate reaction and analyzed by direct sequencing of both strands to confirm and characterize the nature of the mutations.

DNA extraction and PCR–SSCP analysis. High molecular weight DNA was purified by digestion with proteinase K and extraction in phenol–chloroform. DNA samples (100 ng) were subjected to PCR in a mixture (10 µl) as described previously (19), using two appropriate oligonucleotides as primers. The nucleotide sequences of the primers used were as previously described (19). The PCR mixture was heated to 95°C with an equal volume of formamide dye mixture (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA); 2 µl of the preparation was applied to a 6% polyacrylamide gel, both with and without 10% glycerol. Electrophoresis was performed at 10 W for 12–15 h at room temperature. The gel was dried on filter paper and exposed to X-ray film at –80°C for 1–12 h with an intensifying screen.

Direct DNA sequencing. PCR was performed with 1 µg of genomic DNA, 200 ng of each primer, 200 µM dNTPs, 1x PCR reaction buffer, and 2.5 U Taq polymerase. Aliquots of PCR amplified mixtures were diluted with 2 ml of distilled H2O and spun in a Centricon 30 micro-concentrator (Amicon) to remove excess primers and dNTPs. DNA was then resuspended in 50 µl of 10 mM Tris pH 8.0 and 1 mM EDTA. Direct sequencing of the amplified products followed the instructions of the Promega fmolTM DNA sequencing system technical manual.

Statistical analyses. Statistical analyses were performed with the Statistical Analysis System (SAS) statistical package (version 6.12). The association between the incidence of p53 mutations and age, sex, TNM stage, cigarette smoking, alcohol drinking and BQ chewing was examined by {chi}2 test. Multiple logistic regression was further used to assess the most important variables attributed to the incidence and pattern of p53 mutations.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One hundred and eighty-seven consecutive patients with a diagnosis of OSCC were enrolled in the study. The demographic data of the patients are shown in Table IGo. Advanced stage III or IV cancer was diagnosed in 62.0% (116/187) patients. The most common primary sites were the bucca and the tongue. Eighty-five percent (159/187) of the patients smoked, 54.0% (101/187) were users of alcohol and 76.5% (143/187) chewed BQ.


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Table I. Characteristics of the patients with OSCCs (n = 187)
 
Table IIGo shows the mutations in the p53 gene in the 91 oral tumor samples (48.66%). Among the 187 primary OSCCs examined, nine samples contained two different mutations of the p53 gene. p53 exon 5 contained 34 (34%) of the 100 mutations identified in this series, with 30 (30%) mutations detected in exon 8, 21 (21%) in exon 7, 12 (12%) in exon 6 and 3 (3%) in exon 9. Among the 98 mutations with an identified sequence, 14 (14.29%) were deletions, four (4.08%) were insertions, 14 (14.29%) were non-sense mutations, 64 (65.31%) were missense mutations and two (2.04%) were splice-site mutations; 37 (37.76%) were G:C to A:T transitions and 23 (23.47%) were G:C to T:A transversions (Table IIIGo).


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Table II. Mutations of the p53 gene and clinicopathological parameters in Taiwanese patients with OSCC
 

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Table III. p53 mutations identified in 91 patients with OSCC
 
The incidence of p53 mutations was not associated with age, sex, TNM stage, cigarette smoking or BQ chewing. However, alcohol drinkers had a significantly higher incidence (57/101, 56.44%) of p53 mutations than non-users (34/86, 39.53%) (P = 0.02). We further analyzed the interactions of cigarette smoking, BQ chewing and alcohol drinking on the incidence of p53 mutation in these patients with oral cancer. However, because of the small number of females with OSCCs (n = 11) most of who were non-smokers (n = 9), non-drinkers (n = 9) or non-chewers (n = 7), female patients were excluded from this analysis. The effect of alcohol on the incidence of p53 mutations was seen in BQ users with and without smoking habit (Table IVGo), and remained statistically significant (RR = 2.24; 95% CI, 1.21–4.15) after adjustment for cigarette smoking and BQ chewing.


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Table IV. Incidence of p53 mutations in OSCCs: correlation with cigarette smoking, BQ chewing and alcohol drinking in men
 
A specific pattern of mutation was observed in exons 5–9 of the p53 gene in OSCCs from smokers, alcohol users and BQ chewers (Table VGo). G:C to A:T transitions were the predominant mutations observed and associated with BQ and tobacco use. Alcohol drinking enhanced this transition and patients who were both BQ users and alcohol drinkers had the highest incidence. After adjustment for cigarette smoking and BQ chewing, alcohol drinking still showed an effect on G:C to A:T transitions (RR = 2.41; 95% CI, 1.01–5.74). Seventeen of the 18 (94.44%) frameshift mutations including deletions and insertions occurred in smokers. Among them, 14 patients (82.35%) were also BQ chewers. In addition, most (20/22, 90.91%) G:C to T:A transversions occurred in smokers. All A:T to T:A and G:C mutations (n = 11) occurred in BQ chewers. All G:C to C:G transversions occurred in either smokers or BQ chewers. All of the mutations identified in patients with OSCCs in this series were somatic and not germ-line in origin, as DNA from normal tissue adjacent to p53-mutated tumor was negative for p53 mutations by both PCR–SSCP and DNA sequencing analysis.


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Table V. Pattern of p53 mutations in OSCCs: correlation with cigarette smoking, BQ chewing and alcohol drinking in men
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The importance of the p53 tumor suppressor gene in the process of carcinogenesis is well established. In particular, a high incidence of p53 mutation has been demonstrated in tobacco-related cancers. In western countries, the high incidence (42.56%, 163/383) of p53 mutations in OSCCs is associated with known risk factors, specifically tobacco use and alcohol consumption (2024). However, the incidence (19.89%, 37/186) of p53 mutations was much lower in BQ and tobacco associated tumors in studies from India (25,26) and Taiwan (16). In the present study, 91 of 187 (48.66%) patients with OSCCs exhibited mutations in the conserved exons of the p53 gene. These results are similar to reports from western countries (2024), while different from the findings of two previous studies in India (25,26) and one in Taiwan (16). These discrepancies may be partly a result of the inclusion of patients from different geographic areas and differences in the technique used to analyze the mutations. In the present study, SSCP was performed at 10 W in 6% polyacrylamide gel, both with and without 10% glycerol and manual direct sequencing instead of sequencing with autosequencer.

It is extremely difficult to obtain non-BQ and non-tobacco-associated OSCCs as most male OSCC patients are BQ chewers and/or smokers (6). The independent effect of tobacco, BQ and alcohol use on the incidence and pattern of p53 mutation is thus difficult to determine. An increased effect of alcohol on the incidence of p53 mutations was seen in BQ users with and without cigarette smoking. The effect of alcohol on the incidence of p53 mutations remained statistically significant (RR = 2.24; 95% CI, 1.21–4.15) after adjustment for cigarette smoking and BQ chewing. The effect of alcohol on the incidence of p53 mutations was statistically significant in BQ chewers (RR = 1.60; 95% CI, 1.19–2.15). No relationship was identified between alcohol use and the incidence of p53 mutations in smokers who did not chew BQ (RR = 0.73; 95% CI, 0.36–1.46). These findings suggest that a synergism exists between the use of both BQ and alcohol and the induction of p53 mutations in oral cancer and that alcohol does not potentiate the induction of p53 mutations in OSCCs by tobacco carcinogens. This suggestion is in accordance with results obtained by Lazarus et al. (27), who limited their investigation to OSCCs of the oral cavity. In contrast, Brennan et al. (28) found that alcohol did potentiate the induction of p53 mutations in head and neck squamous cell carcinomas. Our analysis was limited to oral cancer and most (92.61%, 163/176) of the OSCCs were in the oral cavity. Thus, these findings may imply that different molecular mechanisms are involved in tumors at different anatomic sites under the same conditions of carcinogen exposure.

The most prevalent types of p53 mutation found in our study were G:C to A:T transitions, G:C to T:A transversions and frameshift mutations. This is similar to the pattern of p53 mutations observed in oral cavity tumors in other studies (16,2023,25,26). G:C to A:T transitions are the most common mutations observed in lung adenocarcinoma in rodents treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (29,30) and in hamster buccal pouch carcinomas induced by N-methyl-N-benzyl nitrosamine, a potent alkylating carcinogen that is similar to tobacco nitrosamine (31). It has been proposed that methylation and deamination of mutation are responsible for G:C to A:T mutations at CpG dinucleotide. Twenty (62.50%) of the 32 G:C to A:T mutations were at CpG site in men, in the present study. Among them, 15 (75.00%) were smokers. G:C to T:A transversions are attributed to NNK in experimental animal models (32). Previous studies (33,34) have suggested that polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke may be activated to species that form adducts with guanine residues in DNA, inducing predominantly G:C to T:A transversion. These mutation types are similar to those we observed in OSCCs. Studies on benzo[a]pyrene (B[a]P), a representative chemical of PAHs, demonstrated that B[a]P is activated by P450 enzymes to diol epoxide metabolites and also forms covalent adducts at adenine residues to a lesser extent than guanine residues (35). The mutagenic consequence of this adduct was either A->T or A->G base substitution depending on the sequence context (36). It is noteworthy that all A:T to T:A and G:C mutations (n = 11) in the present study were occurred in BQ chewers. Among them, nine (81.82%) were also cigarette smokers. Together with findings from other studies (16,2023,25,26) this strongly suggests an important contributive role of tobacco carcinogens to p53 mutation in this series of Taiwanese patients with OSCCs.

It is interesting to note that alcohol drinking increased the likelihood of G:C to A:T transition and patients who chewed BQ and drank alcohol had the highest incidence of this transition in the present study. After adjustment for cigarette smoking and BQ chewing, alcohol drinking still showed an effect on G:C to A:T transitions (RR = 2.41; 95% CI, 1.01–5.74). This raises the possibility that alcohol may enhance the mutagenic effects of betel quid. Safrole–DNA adducts were detected in 77% (23/30) of the OSCC tissues in a study of Taiwanese oral cancer patients with a BQ chewing history (37). Although the major safrole–DNA adduct was characterized as guanine adduct, safrole can also covalently bind to adenine residues in vitro (37). In addition, a similar incidence and pattern of p53 gene mutation was observed among BQ chewers with and without smoking in our analysis. Further study is needed to determine whether safrole or other carcinogens present in the BQ may have a similar pattern of mutagenesis.


    Acknowledgments
 
This study was supported by Grant NSC 89-2314-B182-096 from the National Science Council and Grant DOH88-HR-802 and NHRI-GT-EX89P802P from the National Health Research Institute, Department of Health, The Executive Yuan, Republic of China.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 19, 2001; revised May 29, 2001; accepted May 30, 2001.