p53 Codon 72 and p21 Codon 31 Polymorphisms in Prostate Cancer

The p53 tumor suppressor gene (p53), located on chromosome 17p13, is one of the most commonly mutated genes in all types of human cancer (1, 2). In prostate cancer, mutations of this gene are frequently seen in both germ line and somatic forms (3, 4). p53 protein exhibits a common polymorphism at amino acid 72, resulting in either a proline residue (CCC) or an arginine residue (CGC) (5). An in vitro study has indicated that the p53 Arg/Arg genotype induces apoptosis with faster kinetics and suppresses transformation more efficiently than the Pro/Pro variant (6), suggesting subjects with Pro/Pro variant may be more susceptible to have cancer, probably including prostate cancer, than those with Arg/Arg genotype. However, Henner et al. (7) studied the association between this polymorphism and prostate cancer risk in 115 patients with prostate cancer and 181 controls, all Caucasians. They found that subjects with Pro/Pro, in contrast, had a significantly reduced risk of prostate cancer compared with those with Arg/Arg [odds ratios (OR), 0.14; 95% confidence interval (CI), 0.03-0.71, P = 0.017], which may be in conflict with the results from the in vitro study (6, 7). This conflicting finding could be due to the limitation of the controls not closely conforming to Hardy-Weinberg equilibrium, suggesting probable selection bias, in the study of Henner et al. (7).

Benign prostatic hyperplasia (BPH) is considered a phenotypic manifestation of loss of control in the proliferation of prostate epithelial and/or stromal cells (8). Because p53 plays an important role in the control of the cell cycle, it may be involved in the pathogenesis of BPH. Indeed, expression and mutation of p53 in BPH has been reported (9, 10); however, thus far, no one has investigated the effect of p53 codon 72 polymorphism on the development of BPH.

p21 (Waf1/CIP1), a cyclin-dependent kinase inhibitor that regulates the cell cycle, is a downstream mediator of p53 tumor suppression (11). p21 is induced by the p53 gene, thereby directly mediating p53-induced G₁ arrest. Expression of p21 occurs in prostate cancer and has been associated with the clinical outcome of prostate cancer (12-16), suggesting that p21 may be an important cell cycle regulator involved in carcinogenesis and the progression of prostate cancer.

Gene alterations at p21 might be able interrupt the p53-mediated pathway of cell cycle arrest and increase susceptibility for cancer. A polymorphism in the p21 codon 31 has been found which produces a C-to-A change, causing a substitution from serine (Ser) to arginine (Arg) (17). A series of epidemiological studies found that p21 codon 31 polymorphism was associated with increased risks of lung cancer, cervical cancer, breast cancer, esophageal cancer, and nasopharyngeal cancer (18-23), although conflicting findings were still reported (24, 25). In addition, to our knowledge, no study has examined the effect of p21 codon 31 polymorphism on prostate cancer risk.

Because the gene alterations at p21 also influence the control of cell cycle, we propose that the p21 codon 31 polymorphism might also be involved in the susceptibility of BPH. Therefore, we conducted a case-control study to investigate the association of p53 codon 72 polymorphism and p21 codon 31 polymorphism with prostate cancer and BPH risks in a Taiwanese population.

Patients with prostate cancer were recently diagnosed, between December 2000 and August 2003, and pathologically proven to have prostate adenocarcinoma at the Kaohsiung Medical University Hospital and the Kaohsiung Veterans General Hospital, two medical centers in the city of Kaohsiung in southern Taiwan. During this period, 223 patients with prostate cancer were diagnosed in these two medical centers and 200 (89.7%) of them agreed to participate in this study. Every effort was made to enroll 1 to 2 control subjects that matched each case patient in age (± 3 years) from a population of healthy men who received general health examinations in the two medical centers. If eligible controls were not available from the two medical centers, we attempted to recruit potential controls from the Kaohsiung Municipal United Hospital during the same period. The control subjects did not have significant voiding symptoms (American Urological Association symptom score < 8) (26), their prostate-specific antigen (PSA) levels were within the normal limit (< 4 ng/mL), and they had no history of prostate surgery or clinical signs of prostate hyperplasia or prostate cancer during digital rectal examination. Those who had other known malignancies were excluded.

We recruited a group of symptomatic BPH from the Kaohsiung Medical University Hospital with the criteria of significant lower urinary tract symptoms (American Urological Association symptom score ≥ 8) and prostate enlargement during digital rectal examination and ultrasonography (> 20 gm) (27, 28). Prostate volume was estimated using an ellipsoid formula (29). Those with prostate-specific antigen levels above 4 ng/mL received prostate biopsies to rule out malignancy. Finally, a total of 200 patients with prostate cancer, 181 with BPH, and 247 male controls were included in this study.

This study protocol was approved by the Institutional Review Board of the Kaohsiung Medical University Hospital. After informed consent was obtained, blood samples were collected and all subjects were asked to complete a structured questionnaire. Subjects who had smoked more than 10 cigarettes per week for at least 6 months were defined as smokers, those who had regularly chewed betel quid for at least 6 months were defined as betel quid chewers, and those who consumed beer, wine, or distilled spirits more than once a week for at least 6 months were defined as alcoholic beverage drinkers.

Based on criteria outlined by the American Joint Committee on Cancer tumor-node-metastasis classification system (American Joint Committee on Cancer Staging Manual, 5th edition, 1997), disease stage was determined by pathologic findings, pelvic computed tomography or magnetic resonance image, and radionucleotide bone scans. Pathologic grade was recorded as the Gleason score (30) and classified into three groups: well-differentiated (Gleason score 2-4), moderately differentiated (Gleason score 5-6), and poorly differentiated (Gleason score 7-10).

DNA was extracted from whole blood using a Purgene DNA Isolation Kit (Gentra System, Inc., Minneapolis, MN, USA). p53 codon 72 polymorphism was determined using PCR-RFLP as described in a previous study (31). Briefly, ∼150 ng DNA sample was amplified with two primers: 5′-TTG CCG TCC CAA GCA ATG GAT GA-3′ (forward) and 5′-TCT GGG AAG GGA CAG AAG ATG AC-3′ (reverse; Perkin-Elmer, Taipei, Taiwan). Amplification was performed by initial denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 40 seconds, 68°C for 30 seconds, 72°C for 40 seconds, and a final extension at 72°C for 10 minutes. The PCR product was digested using five units of BstUI (New England Biolabs, Beverly, MA). When BstUI restriction site (Arg allele) was present, the 199-bp fragment was digested into two 113-bp and 86-bp fragments. The Pro allele was not cleaved by BstUI, and had a single band of 199 bp. The heterozygous genotype (Arg/Pro) had three bands (199, 113, and 86 bp; Fig. 1).

Polymorphism in p21 codon 31 was also determined by PCR-RFLP (32). Approximately 150 ng of DNA sample were amplified with two primers: 5′-GTC AGA ACC GGC TGG GGA TG-3′ (forward), and 5′-CTC CTC CCA ACT CAT CCC GG-3′ (reverse; Perkin-Elmer). Amplification was performed by initial denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 40 seconds, 60.3°C for 30 seconds, 72°C for 40 seconds, and a final extension at 72°C for 10 minutes. Final PCR product was digested by BlpI. The Ser alleles with BlpI site generated two 89-bp and 183-bp fragments. The Arg allele which lacked a BlpI site yielded a 272-bp fragment (Fig. 1).

All genotypes were confirmed by direct sequencing at the start of genotyping (Fig. 1). We included one positive control and one negative control sample in each genotyping set (~10 samples). The positive control sample was included to confirm complete digestion of the PCR product by restriction enzymes. The negative control was placed with the same reagents as those used with actual samples with the exception of DNA templates.

Distribution of demographic characteristics and substance use was examined using χ² statistics. For genotypes, the Hardy-Weinberg equilibrium tests were analyzed first. The associations between diseases and genotypes were assessed by calculating the OR and 95% CI. The Statistical Package for the Social Sciences software version 10.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. A two-sided P value < 0.05 was considered statistically significant.

The frequencies of p53 Pro/Pro and p21 Arg/Arg among the general male population in Taiwan were 21.0% and 22.7%, respectively (33, 34). Assuming detection of a significantly increased risk of 2.0-fold in the group with variant genotypes of p53 Pro/Pro and p21 Arg/Arg, the present study had at least 80% power to detect the two-sided 0.05 level of significance in 200 cases and 200 controls.

The demographic and clinical characteristics of prostate cancer cases, BPH and controls are shown in Table 1. The mean age (± SD) for patients with prostate cancer, BPH, and controls were 72.2 (± 7.7), 65.8 (± 8.5) and 72.2 (± 6.6), respectively. In the 200 prostate cancer cases, 128 (64.0%) cases were from Kaohsiung Veterans General Hospital, and 72 (36%) cases were from the Kaohsiung Medical University Hospital. The demographic characteristics, including height, weight, body mass index, education level, cigarette smoking, alcohol drinking, and areca chewing were not significantly different among case, BPH, and control patients. Out of 200 cancer cases, 96 (48.5%) were localized, and the histologic findings of 178 cancer cases (89.0%) were moderately or poorly differentiated.

Table 2 shows the frequencies of p53 and p21 genotypes in prostate cancer cases, BPH, and controls. Frequencies of Arg/Arg, Arg/Pro and Pro/Pro in p53 among controls were 84 (34.0%), 109 (44.1%), and 54 (21.9%), respectively. The frequency of the Pro alleles was 56.1%. Frequencies of the Ser/Ser, Ser/Arg and Arg/Arg in p21 among controls were 76 (30.8%), 119 (48.2%), and 52 (21.1%), respectively. Frequency of the Arg alleles was 45.1%. The distribution of different genotypes in both p53 and p21 polymorphisms among 247 controls closely conformed to expected Hardy-Weinberg frequencies (χ² = 1.389, df = 2, P = 0.499; χ² = 0.074, df = 2, P = 0.964, respectively).

In the prostate cancer cases, the frequencies of p21 Ser/Ser, Ser/Arg and Arg/Arg were 52 (26.0%), 85 (42.5%), and 63 (31.5%); in the BPH patients, they were 48 (26.5%), 82 (45.3%), and 51 (28.2%); and in controls, they were 76 (30.8%), 119 (48.2%), and 52 (21.1%), respectively. The frequencies between the cases and controls were significantly different (χ² = 6.347, df = 2, P = 0.042). After adjusting for other potential covariates (age, body mass index, educational level, cigarette smoking, alcohol drinking, and areca chewing), subjects who carried Arg/Arg were found to have a 1.78-fold increased risk (95% CI, 1.06-3.01, P = 0.029) of developing prostate cancer compared with those who carried Ser/Ser. In the BPH group, subjects who carried Arg/Arg were also found to have an increased risk of developing BPH compared with those carrying Ser/Ser, although this difference did not reach statistical significance (OR, 1.79; 95% CI, 0.97-3.29, P =0.062).

Frequencies of Arg/Arg, Arg/Pro and Pro/Pro in p53 were 66 (33.0%), 92 (46.0%), 42 (21.0%) in the prostate cancer cases, 62 (34.3%), 77 (42.5%), 42 (23.2%) in the BPH patients, and 84 (34.0%), 109 (44.1%), 54 (21.9%) in the controls, respectively. There were no significant differences in the frequencies among the cases and control patients (χ² = 0.158, df = 2, P = 0.924). After adjusting for other potential covariates, subjects who carried Pro/Pro were not associated with increased risk of developing prostate cancer (OR, 1.01; 95% CI, 0.60-1.71, P = 0.960) or BPH (OR, 0.96; 95% CI, 0.53-1.74, P = 0.900) compared with those with Arg/Arg (Table 2). The frequencies of p53 and p21 genotypes between prostate cancer and BPH were not significantly different (χ² = 0.510, df = 2, P = 0.775 for p53; and χ² = 0.531, df = 2, P = 0.767 for p21, respectively).

When dichotomized by median age of 72 years in the prostate cancer cases, the association of p21 Arg/Arg and prostate cancer risk was found to be slightly increased among subjects who were 72 years old and below (Arg/Arg versus Ser/Arg and Ser/Ser: OR, 2.13; 95% CI, 1.16-3.92, P = 0.015; Table 3). In contrast, the effect of p21 Arg/Arg did not reach statistical significance among subjects over 72 years old (OR, 1.45; 95% CI, 0.76-2.77, P = 0.258). However, no significant synergistic effect was noted between age (≤ 72 versus > 72) and p21 polymorphism (Arg/Arg versus Ser/Arg and Ser/Ser; P = 0.566). The above results for the association between p53 polymorphism and prostate cancer risk remained insignificant (data not shown).

Table 4 shows the association between genotypes and prostate cancer risk stratified by disease stage and pathologic grade. The effect of p21 Arg/Arg on prostate cancer risk was found to be significant for localized disease and marginally significant for locally advanced disease (Arg/Arg versus Ser/Arg and Ser/Ser: OR, 1.96; 95% CI, 1.15-3.35 and OR, 1.91; 95% CI, 1.00-3.67). No significant results were found in bone metastasis disease of prostate cancer. Stratified by pathologic grade, the p21 Arg/Arg genotype was found to be associated with significantly increased risk for moderately differentiated prostate cancer (OR, 2.04; 95% CI, 1.17-3.53), but p21 Arg/Arg was not found to be associated with either poorly differentiated or well differentiated prostate cancer. Similar insignificant results were found between p53 polymorphism and clinical/pathological features of prostate cancer (Table 4).

When stratifying the BPH group by the mean prostate volume of 49.8 mL (about 50 mL) as the cut point (> 50 mL versus ≤ 50 mL) and comparing it with that of the controls, we found a significant association between p21 Arg/Arg and large prostate volume (> 50 mL) of BPH risk (Arg/Arg versus Ser/Ser: OR, 2.29; 95% CI, 1.07-4.88; Table 5), although no similar significance was present in small prostate volume (≤ 50 mL) of BPH risk. Again, no significant association was noted between p53 polymorphism and different volumes of BPH risk.

Few studies have investigated the effect of p53 codon 72 polymorphism on prostate cancer risk (7, 35). In the study of a small subset of 28 Japanese men with prostate cancer and 56 controls, Wu et al. (35) found no association between prostate cancer and p53 codon 72 polymorphism (χ² = 0.448, P = 0.799). However, due to the small sample size, the definite insignificant results could not be drawn. Subsequently, in a case-control study of 115 patients with prostate cancer and 181 male controls, Henner et al. (7) found men with Pro/Pro to have a significantly lower risk of prostate cancer (OR, 0.14; 95% CI, 0.03-0.71, P = 0.017) than those with Arg/Arg. However, there may have been selection bias in the control subjects in their study, with the distribution of p53 codon 72 genotypes violating the rule of Hardy-Weinberg equilibrium. In the present study, the frequency of p53 codon 72 genotypes among the 247 male controls closely conformed to Hardy-Weinberg equilibrium, although we did not find any significant associations between p53 codon 72 polymorphism and disease risk of prostate cancer or BPH. Our findings suggest that p53 codon 72 polymorphism may not influence the development of prostate cancer or BPH.

For p21 codon 31 polymorphism, the present study found that subjects with Arg/Arg had a 1.78-fold increased risk of developing prostate cancer compared with those with Ser/Ser. The increased risk was slightly stronger (2.13-fold) in men aged 72 years old and younger. To our knowledge, our study is the first to show a significant association between p21 codon 31 polymorphism and prostate cancer risk.

Numerous studies have suggested that p21 is involved in the carcinogenesis and progression of prostate cancer. Several immunohistochemical studies have shown the expression of p21 to be associated with the clinical outcome of prostate cancer and the progression to androgen-independent prostate cancer (12-16). In a clinical study of prostate cancer specimens, Facher et al. (36) found that 9 (16.7%) of 54 prostate cancer samples had p21 Arg alleles, a proportion that was significantly higher than that found in the 10 (9.1%) of 110 controls.

The p21 codon 31 variant lies within the DNA-binding zinc finger motif (37, 38), suggesting that this polymorphism may encode functionally distinct proteins, but the exact mechanism of how p21 codon 31 polymorphism affects prostate cancer remains unclear. A transfection study in a lung cancer cell line has showed no significant difference between the tumor suppressor abilities of the p21 Ser and Arg alleles (39). Also, in vitro studies using CDK-cyclin kinase assays have reported that both p21 Ser and Arg alleles have similar growth-inhibitory abilities (25, 40). Alternatively, the significant effect of p21 codon 31 polymorphism in the present study may be due to a linkage with other potentially functional polymorphisms that were not examined in this study. Mousses et al. (17) reported that p21 Ser to Arg substitution at codon 31 is strong linkage with p21 C-to-T transition located in 20 bp downstream from the stop codon (the 3′-untranslated region site). Recently, Kibel et al. (41) studied the p21 C-to-T polymorphism in the 3′-untranslated region among 96 patients with prostate cancer and 106 male controls in the United States. They found subjects with CT and TT genotypes to have a significantly higher risk of advanced prostate cancer than those with CC genotype (OR, 2.24; 95% CI, 1.02-4.95). Interestingly, this significant finding was strengthened (OR, 2.33; 95% CI, 0.92-5.96) in a younger age group (≤ the median age at diagnosis of 66 years). Because p21 “CT and TT” genotypes have been closely linked with p21 Arg allele at codon 31, our results were similar to the findings of Kibel et al. (41), although we did not find the significant synergistic interaction between age (≤ 72 versus > 72) and p21 polymorphism (Arg/Arg versus Ser/Arg and Ser/Ser; P = 0.566). Ours and the Kibel study were molecular epidemiologic studies. Future studies need to clarify the functional effects of p21 polymorphisms in codon 31 and the 3′-untranslated region on prostate cancer risk.

The present study also found p21 Arg/Arg to be significantly associated with localized/locally advanced prostate cancer, but not with prostate cancer with bone metastasis (Table 4). Also, this significant association was noted in moderately differentiated disease (OR, 2.04; 95% CI, 1.17-3.53), but not in poorly differentiated disease (OR, 1.49; 95% CI, 0.85-2.61). Although a correlation has been found between expression of p21 and clinical severity of prostate cancer (12, 13), how p21 codon 31 polymorphism affects the clinical/pathological features of prostate cancer remains unknown. One explanation was that the number of cases in our study was relatively small in the subclassification analysis; another possible explanation was that other genetic alterations, except p21codon 31 genotypes, may play an important role in the progression and metastasis of prostate cancer. Nevertheless, whether the p21 codon 31 polymorphism had phenotypic effects on prostate cancer deserves further investigation.

A significant association was found between the p21 Arg/Arg and BPH with large prostate volume (> 50 mL). Men with Arg/Arg had a 2.29-fold increased risk of large-prostate volume BPH, compared with those with the Ser/Ser genotype (Table 5). BPH is characterized by an increased number of epithelial and stromal cells in the periurethral area of the prostate. The increased number of cells may be due either to epithelial and stromal proliferation or to impaired programmed cell death, leading to cellular accumulation (42). p21 is a mediator of the effect of p53 on G₁ arrest in the cell cycle and also interacts with proliferating cell nuclear antigen to control DNA replication (43). Expression of p21 has been reported in BPH (44). Because the alteration p21 gene also interrupts the cell cycle and influences the progression of apoptosis (45), we propose that gene alteration in p21 could cause an imbalance between prostate cell proliferation and apoptosis, and may also participate in the pathogenesis of BPH.

In the present study, patients with BPH, and prostate-specific antigen levels above > 4 ng/mL, were screened for cancer by prostate biopsy to avoid undetectable, occult prostate tumor. Therefore, the misclassification of outcome should be reduced. In conclusion, the p21 codon 31, but not the p53 codon 72, polymorphism was associated prostate cancer risk, especially in younger men (≤ 72 years). p21 codon 31 polymorphism was also associated with disease stage and pathologic grade. In addition, the p21 Arg/Arg was related with the risk of BPH with large prostate volume (> 50 mL). Furthermore, in vitro and in vivo studies are needed to elucidate the functional effect of p21 codon 31 polymorphism on the development of prostate cancer or BPH.