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

Carcinogenesis, Sep 2001

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.

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

Ling-Ling Hsieh 2 3 Pei-Feng Wang 2 I.-How Chen 1 3 Chun-Ta Liao 1 3 Hung-Ming Wang 0 3 Ming-Chi Chen 2 3 Joseph Tung-Chieh Chang 3 5 Ann-Joy Cheng 3 4 0 Division of Hematology/Oncology Department of Internal Medicine , Tao-Yuan , Taiwan 1 Department of Otolaryngology Oncology, Head and Neck Surgery , Tao- Yuan , Taiwan 2 Department of Public Health, Chang Gung University , Tao-Yuan , Taiwan 3 Taipei CGMH Head and Neck Oncology Group , Tao-Yuan , Taiwan 4 Department of Medical Technology, Chang Gung University , Tao-Yuan , Taiwan 5 Department of Radiation Oncology, Chang Gung Memorial Hospital , Tao-Yuan , Taiwan To whom correspondence should be addressed Email: - Introduction Characteristic 176 (94.1) 11 (5.9) 174 (93.05) 3 (1.60) 55 (29.41) 9 (4.81) 69 (36.90) 18 (9.63) 7 (3.74) 13 (6.95) 6 (3.21) 7 (3.74) 24 (12.8) 47 (25.1) 26 (13.9) 90 (48.1) 159 (85.0) 101 (54.0) 143 (76.5) Case ID Age (year) Smoking BQ chewing Drinking TNM Nucleotide change TGCTAC TGCTTC TGCTTC TGCTAC TGCTTC Amino acid change CysTyr CysPhe CysPhe CysTyr CysPhe CysPhe Truncated GlnPro ValAsp Truncated Truncated ProLeu ProLeu ProLeu ProLeu GlyVal Truncated ValPhe ValPhe CGTT ArgPhe CT AlaVal GC MetIle GC MetIle GA AlaThr CT GlnStop Del 8 Truncated TC ValAla GA ArgHis GA ArgHis GA ArgHis GT CysPhe GT CysPhe GA CysTyr Ins 1 Truncated CA ProThr TC IleThe Case ID Age (year) Smoking BQ chewing Drinking TNM Nucleotide change ATCAT CGATGA TTGTAG TTGTAG GATGTT AGAA ACTT TTTTT CATCGT ATGATA ATGGTG TGTCGT CGTTGT CGTCAT CGTCAT TGTTTT TGTTTT AGAACA CGGTGG CGGGGG ACAAC 285 GAGCTTTGAG...GAGGAA 287 GAGTAG 287 GAGTAG 290 CGGGAGA . . . AATCTCGC 291 AAGTAG 294 GAGTAG 294 GAGGG 298 GAGTAG 298 GAGTAG 306 CGATGA 306 CGATGA 308 CTGTG 317 CAGTAG 317 CAGTAG 3 intron GgtaaGctaa 3 intron GgtcGttc Amino acid change Truncated ArgStop LeuStop LeuStop AspVal Truncated Truncated Truncated HisArg MetIle MetVal CysArg ArgCys ArgHis ArgHis CysPhe CysPhe ArgThr ArgTrp ArgGly Truncated Truncated GluStop GluStop Truncated LysStop GluStop Truncated GluStop GluStop ArgStop ArgStop Truncated GlnStop GlnStop Affect splicing Affect splicing Materials and methods Patients, tissue specimens and clinical diagnosis 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 59 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. Direct DNA sequencing. PCR was performed with 1 g of genomic DNA, 200 ng of each primer, 200 M dNTPs, 1 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 l of 10 mM Tris pH 8.0 and 1 mM EDTA. Direct sequencing of the amplified products followed the instructions of the Promega fmol DNA sequencing system technical manual. 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 I. 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. Table II shows the mutations in the p53 gene in the 91 oral tumor samples (48.66%). Among the 187 primary OSCCs Base-pair change GCAT GCTA GCCG TAAT ATCG ATGC Deletion Insertion p53 mutationsa No. identified Percent of total aA total of 100 mutations were identified in tumor tissues from 91 patients, two mutations were not successfully sequenced. RR (95% CI) Cigarette smoking BQ chewing Alcohol drinking Tumors tested Mutations detected (%) 43 (57.33) 15 (29.41) 6 (100.00) 4 (57.14) 7 (43.75) 9 (60.00) 0 (0.00) 1 (25.00) Discussion ) .)126 .)500 .)250 .)500 .)286 .)000 .0000 (3 (2 (6 (2 (4 (3 (1 15 4 5 1 3 3 0 1 23 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. (...truncated)


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Ling-Ling Hsieh, Pei-Feng Wang, I.-How Chen, Chun-Ta Liao, Hung-Ming Wang, Ming-Chi Chen, Joseph Tung-Chieh Chang, Ann-Joy Cheng. Characteristics of mutations in the p53 gene in oral squamous cell carcinoma associated with betel quid chewing and cigarette smoking in Taiwanese, Carcinogenesis, 2001, pp. 1497-1503, 22/9, DOI: 10.1093/carcin/22.9.1497