Genetic Polymorphisms of p 53 Codon 72 and Bladder Cancer Susceptibility: A Hospital-Based Case-Control Study

Abstract

Background: p53 is one of the most widely investigated molecular markers in bladder cancer and its polymorphisms have been related to individual cancer risks. The objective of this study was to explore the association of p53 codon 72 polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population. Methods: We investigated the impact of p53 codon 72 polymorphism in a hospital-based case-control study of bladder cancer. We tested peripheral blood samples from 120 patients with bladder cancer and 120 healthy individuals of similar age and from the same geographical region. The polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. Results: There was an association between smoking status and bladder cancer (odds ratio [OR] = 2.25; 95% confidence interval [95% CI] = 1.31, 3.87; p = 0.003). Patients with bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.53; 95% CI = 0.31, 0.89; p = 0.02) and Arg allele (OR = 0.66; 95% CI = 0.45, 0.95; p = 0.03) than controls. Patients with invasive bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.29; 95% CI = 0.10, 0.88; p = 0.03) than those with superficial bladder cancer. When stratifying by the grade and histological type of bladder cancer, we found no statistical association. Conclusions: These data suggest that the p53 codon 72 Arg/Arg genotype and Arg allele are associated with a lower risk of bladder cancer in Chinese population.

Introduction

Bladder cancer is the fourth most common cancer in men and the ninth most common cancer in women in the United States. In 2008, there were 69,000 newly diagnosed bladder cancer and 14,000 deaths (Jemal et al., 2008). In 2002, about 357,000 cases of bladder cancer occurred throughout the world, making it the ninth most common cause of cancer for both men and women. Risk factors of bladder cancer could be classified into three types: chemical or environmental exposures, genetic and molecular abnormalities, and chronic irritation (Williams and Stein, 2004; Bryan et al., 2005a, 2005b; Kaufman et al., 2009). About 70% of all newly diagnosed cases of bladder cancer present as superficial tumors, about 50%-70% of those superficial tumors will recur, and ∼10%-20% will progress to muscularis propria invasive bladder cancer (Rubben et al., 1988). It remains a challenge to predict which cases will progress from superficial tumors to muscularis propria invasive bladder cancer. Some genetic susceptibility factors had been studied in relation to bladder cancer (Sorensen et al., 2004; Mitra et al., 2006; Wallerand et al., 2008).

Tumor suppressor p53 plays important roles in cell-cycle regulation, apoptosis, and DNA repair in different cell types including bladder cancer (Khaliq et al., 2000; Rohini et al., 2007; Huang et al., 2008). Since 1987, the polymorphism at codon 72 of the TP53 gene (Pro72Arg; rs1042522) has been known (Matlashewski et al., 1987); however, it is still a matter of controversy that p53 codon 72 polymorphism could be a genetic susceptibility factor for cancer (Katiyar et al., 2003). The p53 codon 72 polymorphisms contain three genotypes: proline homozygotes (Pro/Pro), arginine homozygotes (Arg/Arg), and heterozygotes (Pro/Arg) (Matlashewski et al., 1987). It was reported that p53 codon 72 polymorphisms were associated with gastric cancer (Zhou et al., 2007), esophageal cancer (Lee et al., 2006), colorectal cancer (Koushik et al., 2006), lung cancer (Matakidou et al., 2003), breast cancer (Tommiska et al., 2005), and cervical cancer (Klug et al., 2009).

Although there are several studies to test the association between p53 codon 72 polymorphism and risk of bladder cancer, results have been inconsistent (Chen et al., 2000; Toruner et al., 2001; Soulitzis et al., 2002; Mabrouk et al., 2003; Horikawa et al., 2008; Murgel de Castro Santos et al., 2009). The objective of this study was to explore the association of p53 codon 72 polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population.

Methods

Study population

A hospital-based case-control study was conducted in the Second Affiliated Hospital of Chongqing Medical University in Southwest China, from March 2008 to March 2010. We tested peripheral blood samples from 120 patients with bladder cancer and 120 healthy individuals of similar age and from the same geographical region. All cases were confirmed by clinical histopathology and staged according to the Tumor Nodes Metastasis (TNM) staging system of the Union Internationale Contre le Cancer. Tumors were graded according to the World Health Organization classification. Information about sex, age, lifetime tobacco exposure of the patient, and histopathology of the tumor was obtained from medical records. Smoking habit was defined as nonsmoker (smoked < 100 cigarettes in lifetime) and smoker. Written informed consent was obtained from all patients and control subjects who participated in this study according to the Declaration of Helsinki. The study had been previously approved by the local ethics committee and the institutional review board of the Second Affiliated Hospital of Chongqing Medical University.

DNA extraction and genotyping

Venous blood specimen was collected from each person after informed consent was obtained. Blood was collected into ethylenediaminetetraacetic acid-containing tubes and the QIAamp DNA blood mini kit (Qiagen, Valencia, CA) was used to extract genomic DNA. Genomic DNA samples were stored at −20°C until use. The p53 codon 72 polymorphisms were analyzed using a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. The following primers were used: 5′-TTGCCGTCCCAAGCAATGGATGA-3′ and 5′-TCTGGGAAGGGACAGAAGATGAC-3′. The reaction mixtures were preincubated for 10 min at 94°C. The PCR conditions were 94°C for 30 s and 55°C for 1 min, followed by 72°C for 1 min for 40 rounds. After confirmation of an amplified fragment of the expected size, 199 bp, on agarose gel, the PCR products were digested with 2 units of restriction enzyme BstUI (New England Biolabs, Beverly, MA) at 60°C for 16 h. The digested PCR products were resolved on a 3% agarose gel and stained with ethidium bromide for visualization under UV light.

Statistical analysis

Associations between the p53 codon 72 genotype frequencies and risks of bladder cancer, tumor stage, grade, and histological type were assessed using the chi-square test or two-sided Fisher's exact test; the odds ratio (OR) with 95% confidence interval (CI) was calculated. Genotype distribution and allele frequencies were compared with the Hardy-Weinberg equilibrium model using the Pearson χ² test. Data were processed using a commercially available software package (SPSS 11.5), with p < 0.05 indicating statistical significance.

Results

Characteristics and risk factors in patients with bladder cancer and controls are listed in Table 1. Cases and controls did not differ in age (p = 0.63) and gender (p = 0.90). There was an association between smoking status and bladder cancer (OR = 2.25; 95% CI = 1.31, 3.87; p = 0.003). Among 120 bladder cancer cases, 85 (70.8%) were superficial (Tis-T1) bladder cancer, 35 (29.2%) were invasive (T2-T4) bladder cancer, 89 (74.2%) were high-grade (G2 + G3) bladder cancer, and 31 (25.8%) were low-grade (G1) bladder cancer. For the histological type of these patients, 97 (80.8%) were papillary bladder cancer and 23 (19.2%) were nonpapillary bladder cancer. Genotype and allele frequencies were in Hardy-Weinberg equilibrium (p = 0.96).

Table 1.

Characteristics and Risk Factors in Patients with Bladder Cancer and Controls

Variations	Bladder cancer (n = 120)	Controls (n = 120)	p
Gender (male/female)	63/57	64/56	0.90
Age (years)	53.7 ± 7.5	53.2 ± 8.6	0.63
Smoking status (smokers/nonsmokers)	54/66	32/88	0.003
Stage (%)
Superficial (Tis-T1)	85 (70.8)
Invasive (T2-T4)	35 (29.2)
Grade (%)
High (G2 + G3)	89 (74.2)
Low (G1)	31 (25.8)
Histological type (%)
Papillary	97 (80.8)
Nonpapillary	23 (19.2)

Patients with bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.53; 95% CI = 0.31, 0.89; p = 0.02) and Arg allele (OR = 0.66; 95% CI = 0.45, 0.95; p = 0.03) than controls (Table 2). Patients with invasive bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.29; 95% CI = 0.10, 0.88; p = 0.03) than those with superficial bladder cancer. When stratifying by the grade and histological type of bladder cancer, we found no statistical association (Table 3).

Table 2.

Genotype and Allele Frequencies of p53 Codon 72 Polymorphism Among Bladder Cancer Patients and Controls

Genotypes	Cases (%)	Controls (%)	OR (95% CI)	p
Arg/Arg	37 (30.8)	55 (45.8)	0.53 (0.31, 0.89)	0.02
Arg/Pro	59 (49.2)	47 (39.2)	1.50 (0.90, 2.51)	0.12
Pro/Pro	24 (20.0)	18 (15.0)	1.42 (0.72, 2.77)	0.31
Arg allele frequency	133 (55.4)	157 (65.4)	0.66 (0.45, 0.95)	0.03
Pro allele frequency	107 (44.6)	83 (34.6)	1.52 (1.05, 2.20)	0.03

CI, confidence interval; OR, odds ratio.

Table 3.

Stratification Analysis of p53 Codon 72 Polymorphism in Bladder Cancer Patients

		Arg/Arg			Arg/Pro			Pro/Pro
Variations	Cases (n = 120)	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p
Stage	120	37 (30.8)	1 (reference)		59 (49.2)	1 (reference)		24 (20.0)	1 (reference)
Invasive	35	4 (14.1)	0.29 (0.10, 0.88)	0.03	20 (57.1)	1.38 (0.65, 2.95)	0.41	11 (31.4)	1.83 (0.79, 4.26)	0.16
Superficial	85	33 (38.8)	1.42 (0.79, 2.55)	0.24	39 (45.9)	0.88 (0.50, 1.53)	0.64	13 (15.3)	0.72 (0.34, 1.52)	0.39
Grade	120	37 (30.8)	1 (reference)		59 (49.2)	1 (reference)		24 (20.0)	1 (reference)
High	89	32 (36.0)	1.26 (0.70, 2.25)	0.44	44 (49.4)	1.01 (0.58, 1.75)	0.97	13 (14.6)	0.68 (0.33, 1.43)	0.31
Low	31	5 (16.1)	0.43 (0.15, 1.21)	0.11	15 (48.4)	0.97 (0.44, 2.14)	0.94	11 (35.5)	2.20 (0.93, 5.20)	0.07
Histological type	120	37 (30.8)	1 (reference)		59 (49.2)	1 (reference)		24 (20.0)	1 (reference)
Papillary	97	28 (28.9)	0.91 (0.51, 1.64)	0.75	49 (50.5)	1.06 (0.62, 1.80)	0.84	20 (20.6)	1.04 (0.53, 2.02)	0.91
Nonpapillary	23	9 (39.1)	1.44 (0.57, 3.63)	0.44	10 (43.5)	0.80 (0.32, 1.95)	0.62	4 (17.4)	0.84 (0.26, 2.71)	0.77

Discussion

In this study, we examined the prevalence of p53 codon 72 polymorphism in a Chinese group of bladder cancer patients and controls. We found that there was a significant statistical association in the distribution of p53 codon 72 polymorphism between bladder cancer patients and healthy individuals. Patients with bladder cancer had a significantly lower frequency of Arg/Arg and Arg allele than controls. Patients with invasive bladder cancer had a significantly lower frequency of Arg/Arg than those with superficial bladder cancer. Our data suggest that the p53 codon 72 Arg/Arg genotype and Arg allele are associated with a lower risk of bladder cancer in a Chinese population.

We found a significant statistical association between smoking status and bladder cancer. Cigarette smoking appears to be the most significant environmental risk factor (Pashos et al., 2002). The systematic literature review conducted by Zeegers et al. found that there was convincing evidence that cigarette smoking status, frequency, and duration substantially increased the risk of bladder cancer (Zeegers et al., 2000, 2004). However, the evidence was unclear for other forms of smoking. A small increased risk for cigar, pipe, and environmental smoking was only possible (Zeegers et al., 2004). Aminobiphenyls in tobacco have been implicated in bladder cancer etiology in smokers (Probst-Hensch et al., 2000). Many people are exposed to the same risk factors, but only part of these persons incur bladder cancer, which shows variation in individual susceptibility to bladder cancer (Manolio et al., 2008; Rahim et al., 2008).

There is accumulating genetic evidence on the susceptibility and clinicopathologic characteristics of bladder cancer. A meta-analysis of 37 case-control studies involving 16,152 subjects conducted by Zeng et al. (2010) found that glutathione S-transferase T1 null status was associated with a modest increase in the risk of bladder cancer. A meta-analysis of 22 case-control studies involving 7471 subjects conducted by Sun et al. (2010) found that XRCC3 Thr241Met polymorphism was weakly associated with the risk of bladder cancer. The meta-analysis by Wang et al. (2009) found that the XPD Asp312Asn polymorphism was susceptible to bladder cancer development. The HuGE-GSEC review by Kellen et al. (2007) found that the glutathione S-transferase P1 Ile 105Val polymorphism was associated with a modest increase in the risk of bladder cancer.

Some studies have investigated the association between p53 codon 72 polymorphism and other cancers. A pooled analysis of individual data from 49 studies conducted by Klug et al. (2009) found that p53 codon 72 polymorphism was associated with cervical cancer. A pooled analysis of 32 studies involving 19,255 subjects conducted by Dai et al. (2009) found that p53 codon 72 Pro allele was emerging as a low-penetrance susceptibility allele for lung cancer development. A meta-analysis of 12 case-control studies involving 4023 subjects conducted by Zhou et al. (2007) found that the p53 codon 72 polymorphism was associated with gastric cancer among Asians. A meta-analysis of 13 case-control studies involving 4079 subjects conducted by Zhao et al. (2010) found that the p53 codon 72 polymorphism might contribute to esophageal cancer development, especially in Asians. A meta-analysis based on 39 case-control studies conducted by Zhang et al. (2010) found that the p53 codon 72 polymorphism might contribute to breast cancer, especially in Europeans. The systematic review and meta-analysis conducted by Tang et al. (2010) found that the p53 codon 72 polymorphism was not associated with colorectal cancer risk. The meta-analysis conducted by Tandon et al. (2010) showed that current evidence about the prognostic value of p53 in patients with squamous cell carcinoma of the head and neck was inconclusive.

The molecular mechanism of the association between p53 codon 72 polymorphism and bladder cancer remains relatively unclear. The study by Dumont et al. (2003) indicated that the codon 72 polymorphic variants of p53 have markedly different apoptotic potential. They found that the Arg allele induced apoptosis markedly better than did the Pro allele. They also found that one source of this enhanced apoptotic potential was the greater ability of the Arg allele to localize to the mitochondria; this localization was accompanied by release of cytochrome c into the cytosol (Dumont et al., 2003).

In conclusion, this study suggests that the p53 codon 72 Arg/Arg genotype and Arg allele are associated with a lower risk of bladder cancer in a Chinese population. Additional well-designed large studies are required to validate this association in different populations.

Footnotes

Disclosure Statement

The authors declare that no conflicts of interest exist.

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