p53 polymorphism and p21WAF1/CIP1 haplotype in the intestinal gastric cancer and the precancerous lesions

Ya-Guang Xi1,2, Ke-Yue Ding2,3, Xiu-Lan Su4, Da-Fang Chen5, Wei-Cheng You1, Yan Shen2,3 and Yang Ke1,2,6

1 Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, China, 2 Chinese National Human Genome Center, Beijing, China, 3 Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, China, 4 Inner Mongolia Medical College, Huhhot, China and 5 Department of Cell Biology and Human Genetics, Peking University, Beijing, China

6 To whom correspondence should be addressed Email: keyang{at}mx.cei.gov.cn


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of intestinal gastric carcinoma involves several precancerous stages. The environmental factor plays an important role in gastric carcinogenesis, while the host's genetic makeup may influence the susceptibility to cancer. In this study we investigated correlations of the p53 variations at codon 72 and p21WAF1/CIP1 haplotype with the risk of intestinal gastric carcinoma. Forty-eight intestinal gastric carcinoma cases (GC), 96 chronic atrophic gastritis (CAG), 96 intestinal metaplasia (IM) and 96 dysplasia (DYS) controls were enrolled in this study. The p53 codon 72 proline allele carriers were found to be more susceptible to progress to GC than to IM (OR = 2.22, 95%CI = 1.05–4.70, P = 0.038). Patients carrying homozygous p21WAF1/CIP1 haplotype A, which contains the serine at codon 31, the cytidine at the 16th base of the second intron, and the cytidine at the 70th base of the exon 3 were more prone to develop GC than to reach the IM or DYS stage (IM versus GC, OR = 3.35, 95%CI = 1.11–10.15; DYS versus GC, OR = 3.27, 95%CI = 1.09–9.80, P = 0.035). The combination of p53 codon 72 variation with the p21WAF1/CIP1 haplotype further distinguished the risk of GC from IM precancerous lesion (OR = 9.31, 95% CI = 1.77–48.85, P = 0.08). These results suggest that p53 and/or p21WAF1/CIP1 genotype may influence the progression during gastric tumorigenesis.

Abbreviations: CAG, chronic atrophic gastritis; DYS, dysplasia; GC, gastric carcinoma; IM, intestinal metaplasia; SNP, single nucleotide polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tumor suppressor protein p53 plays a critical role in cell cycle control and apoptosis (1). Somatic mutations in p53 are found in >50% of human cancers (2). However, germline mutation in p53 is rare. In addition to mutations, several polymorphisms in p53 have been described. One of the most common variants associated with cancer development is the codon 72 single nucleotide polymorphism (SNP), which results in the substitution of proline for arginine (3). Although the possibility that polymorphic variants in p53 might contribute to cancer susceptibility has been extensively investigated, the issue remains highly complicated. In studies the codon 72 Pro/Pro homozygote was reported to be associated with the risks of a variety of cancers (46). However, controversial results were found (7,8). In addition, codon 72 arginine homozygote in p53 was reported to increase the risk of human papillomavirus-associated cervical cancer (911), although other studies were unable to confirm this correlation (1215). Moreover, the Arg/Arg homozygote at codon 72 was also observed to be associated with other cancers (1619).

One of the notable effectors of p53 is p21WAF1/CIP1, which is a general inhibitor of cyclin-dependent kinases. It functions to negatively control the cell cycle (20,21). p53 up-regulates p21WAF1/CIP1 expression in response to DNA damage leading to cell cycle arrest at the G1 checkpoint (21). Alteration in p53 function results in the loss of p21WAF1/CIP1 expression, although the p53-independent pathways also regulate it (22) In contrast to p53, mutation of p21WAF1/CIP1 is rare (23). Polymorphisms of p21WAF1/CIP1 have been speculated to change its function and the high-risk genotypes have been reported to be associated with different cancers (2428). Gastric cancer is the second most common malignancy worldwide (29). There are two histologically distinct gastric carcinomas (GC), the diffuse-type and the intestinal GC (30). The environmental factor plays an important role in GC formation, especially in intestinal type (31), while host genetic makeup may contribute to the cancer susceptibility (32). The link between p53 polymorphisms and the development of gastric cancer has been studied by several groups (3335). Shepherd et al. analyzed the codon 72 variations in 217 gastric cancer tissues and found that Arg/Arg genotype is more common in Caucasians whereas the proline allele is more common in Blacks. The two variants distributed equally in intestinal and diffuse types (35). Studies performed in Japanese patients did not show a significant difference in p53 codon 72 genotype frequencies amongst the intestinal carcinoma cases and controls (33,34). The pathogenesis of intestinal GC involves a multi-step process. It has been proposed that the sequential changes, namely chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS) occur before the formation of cancer (31). It is possible that p53 and p21WAF1/CIP1 polymorphisms may associate with specific pathological steps of gastric cancer development and contribute to tumor progression. In the present study we analyzed both p53 and p21WAF1/CIP1 polymorphisms in the subjects with the intestinal GC and precancerous lesions. The combination of p53 polymorphism with the p21WAF1/CIP1 haplotype was investigated further.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specimens
The blood samples used in this study were collected from the subjects enrolled in a population-based gastroscopic screening carried out in Linqu county, a gastric cancer high incidence area in China between 1989 and 1990 as described previously (36). Intestinal carcinoma cases and precancerous controls were matched with age and gender. The samples included 48 GC, 96 CAG, 96 IM and 96 DYS. The pathological diagnoses of gastric lesions were made based upon the most severe alteration among any of the biopsies by a panel of three senior pathologists. The quality control slides were confirmed by the reference pathologists from China and the US (37).

p53 polymorphism analysis
DNA was extracted from blood samples by standard procedures. To identify the p53 polymorphism at codon 72, a 550 bp genomic fragment covering codon 72 was amplified by PCR using primers 5'-GAC CTG TGG GAA GCG AAA A-3' and 5'-CCT AAG GGT GAA GAG GAA TC-3'. The reaction was carried out in a 15-µl reaction mixture containing genomic DNA 1 µl, 10x HotStar Taq PCR buffer (Qiagen) 1.5 µl, Q solution for PCR (Qiagen) 3.0 µl, 1.5 mM MgCl2, 0.033 mM concentration of each dNTP, 5 pmol primer and 0.6 U of Hotstar Taq polymerase (Qiagen). The reaction started at 95°C for 15 min. Thirty-five rounds of thermal cycling were followed in the conditions: denaturation at 95°C for 30 s, annealing at 57.5°C for 45 s, extension at 72°C for 35 s and the final extension was carried out at 72°C for 10 min on the Gene-Amp PCR System 9700 (PE Applied Biosystems). PCR products were purified by MultiScreen-PCR purifying plate (Millipore Company USA) and used for sequencing. The dye-terminator sequencing reaction was performed in a total volume 10 µl containing 2 µl of PCR product, 4 pmol of sequencing primer, 3 µl of Bigdye terminator sequencing mix (PE Applied Biosystems). Cycling conditions were as follows: initial denaturing 94°C for 2 min, followed by 30 cycles at 94°C for 10 s, 55°C for 5 s and 60°C for 4 min. After ethanol precipitation, the reaction products were performed for sequencing by ABI 3700 DNA sequencer (PE Applied Biosystems) according to the manufacturer's instructions. The sequencing chromatograms were base-called with Phred (version 0.990722), assembled with Phrap (version 0.990329), scanned by PolyPhred (version 3.5) and the results were viewed with Consed (version 9.0). The PCR and sequencing primers were designed by Primer Premier (version 5.0) software.

p21WAF1/CIP1 polymorphism analysis and haplotype construct
To identify the polymorphisms in p21WAF1/CIP1, a 3.3 kb genomic fragment covering the coding sequence was amplified by PCR and checked by direct sequencing. The primers used in PCR and sequencing reactions were listed in Table I. The reaction started at 95°C for 15 min. Thirty-five rounds of thermal cycling were followed in the conditions: denaturation at 95°C for 30 s, annealing at 56–61.5°C for 45 s, extension at 72°C for 35–100 s and the final extension was carried out at 72°C for 10 min on the Gene-Amp PCR System 9700 (PE Applied Biosystems). Other conditions were the same as above. The PHASE software was used to construct the haplotypes for identified polymorphisms.


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Table I. Primer sets used for PCR amplification and sequencing of p21WAF1/CIP1 gene

 
Statistical analysis
The associations between cases and different controls were analyzed by {xi}2 test for gender, age and genotype. The odds ratio (OR) and 95% confidence intervals (CI) were estimated by logistic regression analysis that was adjusted by gender and age. All analyses were performed with SAS software (SAS version 8.12 software package, SAS Institute, Cary, NC).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The GC and precancerous lesions used in this study were matched for gender and age as shown in Table II. The male/female ratios were 35/13 for GC, 55/41 for CAG, 64/35 for IM and 55/41 for DYS. The mean age was 58.6 years (range = 46.2–71.0) for GC, 54.8 years for CAG (range = 46.9–62.7), 58.5 years for IM (range = 50.9–66.1) and 58.1 years for DYS (range = 50.2–66.0).


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Table II. Distribution of age and gender among cases and controls

 
The genotype distribution of the p53 codon 72 in the cases and controls are summarized in Table III. The distributions of the genotype among cases and all precancerous lesions were in Hardy–Weinberg equilibrium. There was no significant difference in the distributions of p53 genotypes among GC, CAG and DYS. However, the proline allele frequencies between GC and IM showed significant difference (GC versus IM, OR = 2.2, 95%CI = 1.05–4.7, P = 0.038, Table III).


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Table III. Genotype frequencies of p53 gene codon 72 variation among cases and controls

 
Three polymorphisms of p21WAF1/CIP1 were identified. These include codon 31 (AGC->AGA, Serine->Arginine), +460 (C->G, 16th base in the second intron of p21WAF1/CIP1 gene) and +1718 (C->T, 70th base in the exon 3 of p21WAF1/CIP1 gene). The frequencies of the three variations were not statistically different among GC, CAG, IM and DYS. The results are shown in Table IV.


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Table IV. Genotype frequencies of three SNPs in p21WAF1/CIP1 gene among cases and controls

 
The haplotypes with the existing three SNPs in p21WAF1/CIP1 gene were constructed by using PHASE software, which is based on Bayesian algorithm and makes a more reliable estimate than classical EM algorithm (38). A total of seven haplotypes (A, B, C, D, E, F and G) were constructed. Three haplotypes were found in the GC group. All seven haplotypes were found in CAG and six haplotypes existed in both IM and DYS. Haplotype G (Arg-C-C) was only found in the CAG group. The distribution of different haplotypes in each group is shown in Table V. The A, B and C types represented the major haplotypes in the tested subjects. The sum of the A, B and C haplotype frequency corresponded to >93.0% genotypes among all groups (GC 100%, CAG 94.2%, IM 93.6% and DYS 93.6%). The A, B and C haplotypes were then selected to assess the susceptibility to GC. The association analysis and logistic regression analysis showed that only haplotype A was found to have an association with the cancer risk.


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Table V. Distribution of the estimated haplotype frequencies for p21WAF1/CIP1 gene in cases and controls

 
Haplotype A (Ser-C-C) represents the majority genotype in all haplotypes. Its homozygote (haplotype A/A) has a higher frequency in the cases (22.9%) than in the controls (CAG 13.5%, IM 9.4% and DYS 9.4%). When comparison was carried out between cancer cases and different lesions the increased GC risk was found in patients with homozygous haplotype A (IM versus GC, OR = 3.35, 95% CI = 1.11–10.15, P = 0.032; DYS versus GC, OR = 3.27, 95% CI = 1.09–9.80, P = 0.035). The results are summarized in Table VI.


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Table VI. Distribution frequencies of haplotypes in p21WAF1/CIP1 gene among cases and controls

 
We next investigated whether codon 72 polymorphism in p53 would have an additive effect with p21WAF1/CIP1 haplotype A in GC risk. The IM individuals carried both codon 72 proline allele in p53 and p21WAF1/CIP1 haplotype A/A showed significant risk to develop GC (OR = 9.31, 95% CI = 1.77–48.85, P = 0.008) compared with the individuals who only carried codon 72 proline allele in p53 (OR = 2.86, 95% CI = 0.75–10.99, P = 0.125) or p21WAF1/CIP1 haplotype A/A (OR = 4.26, 95% CI = 0.76–23.76, P = 0.099). In addition, no significant difference was found among GC, CAG and DYS. The data are summarized in Table VII.


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Table VII. Combined associations of haplotypes in p21WAF1/CIP1 gene and p53 gene codon 72 variation among cases and controls

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we investigated the p53 polymorphism and p21WAF1/CIP1 haplotype in the intestinal gastric cancer and its precancerous lesions. The subjects were from a population-based gastroscopic screening carried out in a gastric cancer high prevalence area in northern China. The intestinal carcinoma is the major type malignancy among gastric cancer patients there. Among the participants, 98.1% had CAG, 52.8% had IM and 20% had DYS. In addition, among those with GC, concomitant DYS and IM were found. For instance, in the 13 GC cases detected in the screening, eight (62%) also had DYS and the remaining had IM without DYS (37). Therefore, we used different precancerous lesions as the control to determine the role of genotype for GC progression.

We observed that the proline allele carriers have a higher risk of developing the intestinal type of gastric cancer in comparison with the IM risk, although this was not found when compared with CAG and DYS groups. The codon 72 variations in p53 have been speculated to increase the susceptibility to cancers. Extensive investigations were carried out in different cancers. The results were inconclusive as mentioned above. However, p53 polymorphisms combined with either environmental factors, such as smoking, or other genetic factors were more likely to contribute to the risk of cancers (3942). In addition, the p53 polymorphism showed stronger link to cancers when subjects were limited in certain conditions such as disease types, disease stages, age of onset, etc. (4345). Taken together, the data suggest that the codon 72 variation in p53 alone is insufficient to increase cancer risk. Rather, the variation may become sensitive to specific conditions and predict the susceptibility to cancer. In the case of intestinal GC, cancer development is closely associated with the procancerous process. Helicobacter pylori infection results in the chronic inflammation as the initial pathological change of gastric mucosa. Host genetic makeup and other environmental factors may modulate the inflammation process (46,47). A recent study suggests that the interleukin 1 beta gene polymorphisms might determine the outcomes of gastric inflammation (48). Our data suggest that a p53 polymorphism at codon 72 might play a role in promoting GC development from IM but not from GAC or DYS to GC. However, a large size investigation is required to reach a conclusion. It would also be interesting to determine the environmental and/or other genetic factors that interact with p53 polymorphism.

Three previously reported SNPs in p21WAF1/CIP1 (49,50) were found in the subjects enrolled in our study. None of these SNPs alone were associated with either increased risk of gastric cancer or precancerous lesions. Among the three SNPs, the codon 31 polymorphism is the only one located in the coding region. The substitution of serine to arginine has been found in varieties of malignancy (24,25,5162). However, the link between the codon 31 polymorphism and cancer risk was only found in the small portion of these malignancies (51,58,60). The results presented here support that codon 31 polymorphism alone is not sufficient to predict gastric cancer risk. In contrast to individual SNPs, we observed that haplotype A (Ser-C-C) carriers significantly increased the probability of developing gastric cancer. These results suggest that p21WAF1/CIP1 haplotype A, like p53 polymorphisms may play a role in the specific pathological stage and promote the progression to GC. In contrast to p53, the p21WAF1/CIP1 haplotype A affects both IM and DYS groups suggesting that p21WAF1/CIP1 may act in the gastric pathogenesis through both p53-dependent and -independent pathways.

Whether a combination of p53 codon 72 proline allele and p21WAF1/CIP1 haplotype A improves the prediction of gastric cancer risk interested us. When comparison was carried out between cancer cases and different precancerous lesions, the significant difference was only found between IM and GC. In addition, The OR of the interaction is much higher than either p53 proline or p21WAF1/CIP1 haplotype A alone compared with IM and GC. This result suggests that the p53 polymorphism and p21WAF1/CIP1 haplotype are likely to synergistically affect the events to develop GC. p21WAF1/CIP1 is an important effector of p53 in control of cell proliferation. It would be very interesting to investigate the molecular mechanism of p53/p21WAF1/CIP1-mediated cell cycle arrest in the development of GC, particularly in the progression from IM to GC.

The association between p53 and cancer susceptibility has been an interesting issue. Extensive investigations were carried out in different cancers. The results were inconclusive. The genetic variations that might be stabilized among the population are those that cause no pivotal effect in normal condition. However, under special circumstances the variations may predispose the carriers to diseases. Therefore, analysis combined the genetic variations and the other internal and external factors may lead to a better understanding of the role of genetic makeup in cancer pathogenesis.


    Acknowledgments
 
This study is supported by State Key Basic Research Program Grant G1998051203, ‘863’ key project of National Ministry of Science and Technology Grant 2002BA711A06 and Beijing Municipal Science and Technology Commission (to Prof. Yang Ke).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 23, 2004; revised June 25, 2004; accepted June 29, 2004.