ANTICANCER RESEARCH 27: 1559-1564 (2007) Association of p53 and p21(CDKN1A/WAF1/CIP1) Polymorphisms with Oral Cancer in Taiwan Patients DA-TIAN BAU1,4*, MING-HSUI TSAI2*, YEN-LI LO6, CHIN-MOO HSU1, YUHSIN TSAI4, CHENG-CHUN LEE1 and FUU-JEN TSAI3,5 Departments of 1Medical Research, 2Otolaryngology and 3Pediatrics, China Medical University Hospital, Taichung; 4Graduate Institute of Chinese Medical Science and 5College of Chinese Medicine, China Medical University, Taichung; 6Division of Biostatistics and Bioinformatics, National Health Research Institutes, Taiwan, R.O.C. Abstract. Background: The tumor suppressor gene p53 and Oral cancer is one of the most commonly diagnosed cancers its downstream effector p21(CDKN1A/WAF1/CIP1) are in the world, the fourth most common in Taiwan. Its rising thought to play major roles in the development of human incidence and mortality poses a formidable challenge to malignancy. Polymorphic variants of p53, at codon 72, and oncologists. Early premalignant oral lesions, such as CDKN1A, at codon 31, have been associated with cancer leukoplakia, appear as a white patch in the oral cavity of susceptibility, but few studies have investigated their effect on betel and tobacco consumers; five to ten percent progress oral cancer risk. Materials and Methods: In this hospital-based toward malignancy (1). Therefore, the identification of a case-control study, the association of p53 codon 72 and biomarker for screening the high-risk population for CDKN1A codon 31 polymorphisms with oral cancer risk in a increased predisposition to cancer is of utmost importance Taiwanese population were investigated. In total, 137 patients for primary prevention and early anticancer intervention. with oral cancer and 105 age-matched controls recruited from The protein p21(Cdkn1a/Waf1/Cip1), encoded by the the Chinese Medical Hospital in Central Taiwan were CDKN1A locus, is a universal inhibitor of cyclin-dependent genotyped. Results: We found a significant difference in the kinases (Cdks), which suggests its widespread role in frequency of the p53 genotype, but not the CDKN1A genotype, regulating the cell cycle. The human CDKN1A gene consists between the oral cancer and control groups. Those who had of three exons of 68, 450 and 1600 bp (2). In normal cells, Arg/Arg at p53 codon 72 showed a 2.68-fold (95% confidence p21 exists predominantly in quaternary complexes with interval=1.19-6.01) increased risk of oral cancer compared to cyclins, Cdks, and PCNA to inhibit the activity of Cdks and those with Pro/Pro. The distribution of the combination of p53 control the G1- to S-phase transition (3). The CDKN1A codon 72 and CDKN1A codon 31 was different in the oral gene has a p53 transcriptional regulatory motif and cells cancer and control groups. The percentages of three subgroups lacking functional p53 tumor suppressor protein express with the p53 GG homozygote were all higher in the oral cancer very low levels of p21, suggesting that p53 regulates group, and the risky double homozygote, p53/CDKN1A CDKN1A expression directly (4). The expression of p21 GG/CC form, was almost 9-fold higher than the control group. induces differentiation of normal and transformed cells, and Conclusion: Our findings suggest that the homozygous Arg the involvement of p21 in terminal differentiation has been allele of the p53 codon 72 may be associated with the observed in several cell systems (5, 6). Differential development of oral cancer and be a useful marker for primary regulation of p21 by p53 and retinoblastoma has been prevention and anticancer intervention. reported in cellular response to oxidative stress (7). In addition, several recent studies suggest a role for p21 in apoptosis (8). Quercetin-induced apoptosis in hepatocytes was also associated with the regulation of p21 protein *Both authors contributed equally to this work. expression in a p53-independent pathway (9). In view of the central role of p21 in inducing growth Correspondence to: Fuu-Jen Tsai, MD, Ph.D., Department of arrest, terminal differentiation, or apoptosis, alterations in Pediatrics, Medical Genetics and Medical Research, China Medical the CDKN1A gene and its expression may play a vital role in University Hospital, 2 Yuh-Der Road, Taichung, 404 Taiwan, R.O.C. Tel: +88 6422052121 Ext 7080, Fax: +88 6422033295, the pathogenesis of cancer. Alterations in CDKN1A e-mail: [email protected] expression have been observed in a wide variety of human cancers, including ovarian, uterine, cervical, colorectal, Key Words: p53, p21, polymorphism, oral cancer, carcinogenesis. hepatocellular, and head and neck carcinomas (10-12). 0250-7005/2007 $2.00+.40 1559 ANTICANCER RESEARCH 27: 1559-1564 (2007) Currently, knowledge of the biological and clinical relevance categorical variables. Information on these factors was identified of CDKN1A alterations in oral lesions is meager (13, 14). as established habits when occurring at least twice a week for years. Differential expression of p21 protein in tobacco-related Our study was approved by the Institutional Review Board of the China Medical University Hospital and written-informed consent oral tumorigenesis in the Indian population was reported was obtained from all participants. (15). As well known, in response to DNA damage, p21 is a key mediator of the G0-G1 cell cycle arrest induced by p53. Genotyping assays. Genomic DNA was prepared from peripheral Further study revealed that p21 also interacted with PCNA blood leukocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, to cause both G1 and G2 cell cycle arrest in p53-deficient Taiwan) and further processed according to a previous paper (29). cells (5, 16, 17). A novel polymorphism of the CDKN1A Briefly, for p53 codon 72, the primers 5'-TCCCCCTT gene in codon 149 was found in the Indian population and GCCGTCCCAA-3' and 5'-CGTGCAAGTCACAGACTT-3' were used (30), and for CDKN1A codon 31, the primers 5'-GTCAGAA was considered as a marker of genetic susceptibility to CCGGCTGGGGATG-3' and 5'-CTCCTCCCAA CTCATCCCGG- esophageal and oral cancers (18, 19). Bioinformatics 3' were used (31). The following cycling conditions were performed: analysis revealed that the so-called polymorphism of codon one cycle at 94ÆC for 5 min; 35 cycles of 94ÆC for 20 sec, 58ÆC for 149 is not a susceptible site (20). 20 sec and 72ÆC for 20 sec; and a final extension at 72ÆC for 10 min. The p53 gene is a tumor suppressor gene, whose function The PCR products were studied after digestion with BstU1 restriction is partially mediated by transactivating the CDKN1A enzyme for p53 codon 72, or with BlpI for CDKN1A codon 31. promoter which controls the cell cycle and prevents tumor Statistical analyses. To ensure that the controls used were formation (21). It was found that premalignant lesions and representative of the general population and to exclude the squamous cell carcinomas (SCCs) with mutant p53 protein possibility of genotyping error, deviation of the genotype overexpressed p21 (19). p21 immuno-positive well- frequencies of p53 codon 72 or CDKN1A codon 31 single differentiated tumors with p53 missense mutations probably nucleotide polymorphism in the control subjects from those harbor a p21-dependent differentiation pathway activated expected under the Hardy-Weinberg equilibrium was assessed through a p53-independent mechanism (22). Taken using the goodness-of-fit test. Pearson's ¯2 test or Fisher's exact test together, these effects reflect the complexity of the p53/p21 (when the expected number in any cell was less than five) was used to compare the distribution of the p53 and CDKN1A genotypes pathways of cell cycle regulation and differentiation in the between cases and controls. Cancer risk associated with the pathogenesis of oral cancer. Mutations in either p53 or genotypes was estimated as odds ratio (ORs) and 95% confidence CDKN1A were detected in some tumor cells (21, 23) and intervals (CIs) using unconditional logistic regression with polymorphisms of p53 codon 72 or CDKN1A codon 31 were adjustment for age, smoking, alcohol consumption and betel found to be associated with many tumors (24-27). chewing habits. Data was recognized as significant when the Since p53 gene mutations are the most common cancer- statistical p-value was less than 0.05. related genetic alterations, found in ~50% of human cancer (28), and p53 regulates p21 expression (4), we were Results interested in looking for a susceptible site in p53 and CDKN1A genes together in Taiwan population, especially in The mean ages of the oral cancer patients and the controls oral cancer patients. Thus, the main goal of the study was were 53.27 (SD=12.27) and 53.02 (SD=10.08) years, to examine the joint effect of polymorphisms in codon 72 of respectively. The proportion of men and women were the p53 gene and codon 31 of the CDKN1A gene with oral almost the same in the patient and control groups (i.e., 117 cancer in a Central Taiwan population. men and 20 women). Characteristically, the most common sites of primary tumor were the tongue and buccal mucosa Materials and Methods in this Central Taiwan population. Approximately 38% (52 of 137) of the study cohort consisted of patients diagnosed Study population and sample collection. One hundred and thirty- with TNM late stage (III and IV) oral cancer (data not seven patients diagnosed with oral cancer were recruited at the shown). outpatient clinics of general surgery between 1997-2005 at the China Medical University Hospital, Taichung, Taiwan, Republic of The frequency of the alleles for the p53 codon 72 and China. All patients voluntarily participated, completed a self- CDKN1A codon 31 between oral cancer and control groups administered questionnaire and provided peripheral blood samples. is shown in Table I. The Arg allele at p53 codon 72 was One hundred and five non-oral cancer healthy people as controls significantly associated with oral cancer risk (p=0.02).