p53 codon 72 polymorphism (C/G) and the risk of human papillomavirus-associated carcinomas in China

Authors

  • Tao Li,

    1. Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
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  • Zhe-Ming Lu,

    1. Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
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  • Mei Guo M.S.,

    1. Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
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  • Qin-Jiao Wu M.S.,

    1. Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
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  • Ke-Neng Chen M.D.,

    1. Department of Surgery, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
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  • Hai-Ping Xing B.S.,

    1. Anyang Tumor Hospital, Anyang City, People's Republic of China
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  • Qiang Mei B.S.,

    1. Anyang Tumor Hospital, Anyang City, People's Republic of China
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  • Yang Ke M.D., Ph.D.

    Corresponding author
    1. Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, People's Republic of China
    • Laboratory of Genetics, Beijing Institute for Cancer Research, School of Oncology, Peking University, No. 1 Da Hong Luo Chang Street, Beijing, People's Republic of China 100034
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    • Fax: 011-8610-66175832


  • This study was a key project of the Beijing Metropolitan Science and Technology Committee and Human Disease Gene Center at Peking University, Peking, and the Chinese National Human Genome Center, Beijing.

Abstract

BACKGROUND

Human papillomavirus (HPV) plays an important role in the development of carcinomas at various body sites. It was found previously that the p53 codon 72 polymorphism (C/G) is a high-risk factor for the development HPV-associated cervical carcinoma. However, it still was considered controversial in several studies of cervical and esophageal carcinoma.

METHODS

In the current study, the authors used an allele specific polymerase chain reaction (PCR) method to analyze correlation between the p53 codon 72 (C/G) polymorphism and HPV-associated, noncancerous esophageal epithelium as well as esophageal, ovarian, and breast carcinoma in the Chinese population. Esophageal balloon cytology examination samples were obtained from high-incidence and low-incidence populations for esophageal carcinoma in Anyang (Henan Province).

RESULTS

Thirty-six of 48 esophageal balloon samples from the high-incidence population were HPV positive, and 13 of 33 esophageal balloon samples from the low-incidence population were HPV positive. Thirty-nine of 62 esophageal carcinoma samples from Anyang Tumor Hospital were HPV positive. Twenty-six of 39 ovarian carcinoma samples from the Second Affiliated Hospital of Inner Mongolia Medical College were HPV positive. Nineteen of 82 breast carcinoma samples from Beijing Cancer Hospital were HPV positive. It is noteworthy that the distribution of the p53 codon 72 Arg homozygous genotype in HPV positive samples of esophageal epithelium, ovarian carcinoma, and breast carcinoma was significantly higher compared with HPV negative tumor samples. (P < 0.05).

CONCLUSIONS

The current results suggest that the p53 codon 72 Arg homozygous genotype is one of the high-risk genetic factors for HPV-associated malignancies among the Chinese population. Cancer 2002;95:2571–6. © 2002 American Cancer Society.

DOI 10.1002/cncr.11008

Human papillomavirus (HPV) plays an important role in the development of malignant disease at various body sites, including the anogenital area, the upper respiratory tract, the digestive tract, and the breast.1, 2 However, not all HPV-containing lesions proceed to the full malignant phenotype. It has been shown that HPV infection alone may not be sufficient for the malignant transformation of a host cell and requires additional genetic lesions. Germline polymorphisms of some genes involved in multiple steps of carcinogenesis may account for the genetic susceptibility to HPV-associated carcinoma.

The high-risk HPV types, such as HPV type 16 (HPV-16) and HPV-18, encode two viral oncoproteins, E6 and E7, that are expressed in HPV-associated malignancies.1, 2 It has been shown that E7 protein is capable of binding and inactivating the cellular tumor suppressor protein Rb, whereas the E6 protein interacts with and degrades p53 through the ubiquitin pathway. These interactions are responsible for the transforming activity of HPV.3–5 A common polymorphic site in the wild type p53 gene at codon 72 of exon 4 results in translation to either an arginine residue (CGC) or a proline residue (CCC). The frequency of two alleles varies among ethnic groups that dwell at different latitudes.6 The p53 codon 72 polymorphism (C/G) was one of the first susceptible factors of HPV-associated carcinoma that was found in 1998 by Storey et al.,7 who reported that the p53 arginine (p53Arg) isoform increased the susceptibility of p53 to HPV-16 and HPV-18, E6-mediated degradation either in vitro or in vivo. Moreover, there is a significant over-representation of the p53 Arg/Arg genotype in patients with cervical carcinoma compared with the normal population. Individuals homozygous for p53Arg were about seven times more susceptible to HPV-associated carcinogenesis compared with heterozygotes.7 However, many subsequent studies in different populations have failed to prove the results.8–13 The correlation between the p53 codon 72 polymorphism (C/G) and HPV-associated carcinoma remains controversial.

We showed previously that HPV-16 plays a significant role in the pathogenesis of esophageal carcinoma in Anyang, which is recognized as a high-incidence area for esophageal carcinoma in China. Retaining of viral DNA fragments E6/E7 and stable expression of viral genes may be associated with the malignant progression of esophageal epithelial cells.14 Our recent investigation also showed a correlation between HPV and breast and ovarian carcinoma15 (and unpublished results). It was found that the HPV-16 infection rate was significantly higher in samples of epithelial ovarian malignancy (26 of 50 samples; 52%) compared with the rate in normal ovarian tissues (2 of 30 samples; 6.7%), and HPV-16 E6 mRNA was found in 17 of 34 breast carcinoma samples (50%). Based on our previous studies, investigation of genetic cancer susceptibility in the p53 gene, which may be involved in the steps of carcinogenesis, is important for the identification of high-risk individuals in an HPV-infected population.

In the current study, we examined esophageal balloon samples of different histologic grade from the Anyang area and from three types of HPV-associated malignancies to determine the frequency of p53 polymorphism (C/G) in the Chinese population.

MATERIALS AND METHODS

Sample Collection, Preparation, and HPV Detection

Esophageal balloon cytology examination samples were obtained from the Anyang area, which has the highest incidence and mortality of esophageal carcinoma in China. In this area, the esophageal carcinoma-adjusted and age-adjusted mortality rates among 13 counties vary from 141 × 105 population to 23 × 105 population. We collected specimens by esophageal balloon cytology examination from volunteers living in two villages, Shangzhuang and Tiangmiao, which have age-adjusted mortality rates for esophageal carcinoma of 132 × 105 and 52 × 105, respectively. The villages are situated approximately 100 km apart: Shangzhuang is a mountain village, and Tiangmiao is situated in the plain area. Partly because of the natural environment and traffic, the economic conditions in Shangzhuang are poor compared with those in Tiangmiao. Volunteers from Shangzhuang and Tiangmiao were considered high-incidence and low-incidence populations for esophageal carcinoma, respectively. Sample collection and preparation procedures and HPV detection were described previously.14 Briefly, we had the volunteers swallow a deflated balloon, which was then expanded and gently removed, and their esophageal epithelial cells were then collected. Cells on the surface of the balloon were smeared onto slides and sent to the Department of Pathology at Anyang Tumor Hospital for cytologic evaluation. Parallel slides were collected for in situ hybridization of the HPV-16 E6 and HPV-18 E6 genes. After the slide smears were taken, the remaining cells on the balloon were washed off with saline and concentrated by centrifugation at × 4000 g for 5 minutes. The cell precipitation was dissolved in 500 μL lysis buffer (10 mm Tris-Cl, pH 7.8; 100 mm NaCl, 10 mm ethylenediamine tetraacetic acid [EDTA], and 0.5% sodium codicil sulfate [SDS], and 10 μL of 20 mg/mL proteinase K were added to the cell lysate. After incubation at 55 °C overnight, the DNA was extracted with phenol/chloroform. The presence of HPV DNA was evaluated with polymerase chain reaction (PCR) analysis by using type specific primers for the HPV E6 gene of HPV-16, HPV-18, and the HPV E7 gene of HPV-16. The in situ hybridization method was used to detect HPV-16 and HPV-18 E6 mRNA. The single-strand probe was digoxin labeled, and its sensitivity corresponded to the sensitivity of the isotope method. Forty-eight samples were from the high-incidence population, and 36 of those samples were HPV positive. Thirty-three samples were from the low-incidence population, and 13 of those samples were HPV positive. Cytologic evaluation revealed that 17 of 36 samples of normal epithelium were HPV positive, 23 of 34 samples of mild dysplasia were HPV positive, and 8 of 11 samples of severe dysplasia were HPV positive.

In addition, we studied the following samples: There were 62 esophageal carcinoma samples from Anyang Tumor Hospital (39 were HPV positive), 39 ovarian carcinoma samples from the Second Affiliated Hospital of Inner Mongolia Medical College (26 were HPV positive), and 28 breast carcinoma samples from Beijing Cancer Hospital (19 were HPV positive). All of these samples were paraffin embedded archival samples that were collected between 1999 and 2000. HPV detection (HPV-16 and HPV-18) was performed with both PCR and in situ hybridization methods in our previous study.14, 16 We selected 50 healthy blood donors among the Beijing population as a control group. Although our samples were from three different geographic areas, we studied the distribution of the three p53 genotypes in age-matched, healthy control populations in different geographic areas and found no significant difference among these populations (unpublished data). Informed consent was obtained from all individuals.

DNA Extraction

DNA extraction from esophageal balloon cytology samples was described previously.14 DNA from paraffin embedded carcinoma samples was extracted through the following procedures: Five-micrometer, formalin fixed, paraffin sections of carcinoma samples were deparaffinized in xylene; washed with 100%, 95%, and 75% ethanol; pelleted; air dried; and incubated overnight at 55 °C with 10 μL of 20 mg/mL proteinase K in 500 μL lysis buffer (10 mM Tris-Cl, pH 7.8; 100 mM NaCl, 10 mM EDTA, and 0.5% SDS). The DNA was extracted with phenol/chloroform and precipitated with cold ethanol. We extracted DNA from 50 healthy blood donors as follows: First, white blood cells were separated from red blood cells by washing three times in lysis buffer. Then, the DNA was extracted with phenol/chloroform and precipitated with cold ethanol. All DNA samples were dissolved in water and stored at −20 °C. The quality of DNA was confirmed by amplification of each sample with microsatellite primers (D3S1561; GIBCO BRL, Gaithersburg, MD).

Allele Specific PCR Analysis of the p53 Polymorphism

Genotyping of p53 at codon 72 was carried out with an allele specific PCR amplification procedure. Two pairs of primers with different 3′ terminal bases were used to amplify p53 sequences separately (Fig. 1): p53-p1 (GCCAGAGGCTGCTCCCC) and p53-p2 (CGTGCAAGTCACAGACTT) for proline (178 base pairs [bp]) and p53-a1 (TCCCCCTTGCCGTCCCAA) and p53-a2 (CTGGTGCAGGGGCCACG) for arginine (142 bp). The PCR reaction was performed in a total volume of 25 μL. The PCR mixture contained 10 mM Tris-Cl, pH 8.3; 50 mM KCl; 1.5 mM MgCl; 0.01% gelatin; 200 pmol of each primer; 2 U Taq DNA polymerase; and 100 ng DNA. Amplification was performed with denaturation at 94 °C for 2 minutes followed by 30 seconds at 94 °C, 50 seconds at 62 °C, and 30 seconds at 72 °C for 35 cycles, and a final extension for 5 minutes at 72 °C. Then, the PCR products of each sample were mixed and electrophoresed on 2% agarose gels and were visualized with ethidium bromide staining. The samples were renumbered before the p53 PCR assay. Experimenters were blinded to the cytopathologic grade and HPV status of all samples. The PCR assay was reiterated in a different laboratory (located on a different floor of the building) to verify our results. Amplified PCR products were sequenced using both forward and reverse primers (ABI PRISM model 377, version 3.3).

Figure 1.

Detection of p53 codon 72 polymorphism (C/G) by allele specific polymerase chain reaction (PCR) analysis. The structure of the polymorphic site in exon 4 and the primers used for PCR analysis are indicated by arrows. The sequences are described in Materials and Methods.

Statistical Analysis

Statistical analysis was performed by using chi-square tests with SPSS software (version 10.0; SPSS, Inc., Chicago, IL) to determine the differences of p53 codon 72 genotypes among the study groups. P < 0.05 was considered statistically significant.

RESULTS

DNA amplification with microsatellite primers showed that DNA from all specimens was suitable for PCR analysis (data not shown). p53Arg homozygotes and p53 proline (p53Pro) homozygotes were observed as specific bands with the expected sizes of 178 bp and 142 bp, respectively. The heterozygotes were revealed by both bands (Fig. 2). Sequencing results showed that the PCR products were genuine (Fig. 3).

Figure 2.

Allele specific polymerase chain reaction (PCR) amplification of p53 codon 72 polymorphism. Lanes 1 and 9: Marker (100 base-pair DNA ladder; GIBCO BRL). Lanes 3 and 7: p53Pro homozygotes. Lanes 4 and 8: p53Arg homozygotes. Lanes 2, 5, and 6: p53 heterozygotes.

Figure 3.

Sequencing results of p53 polymerase chain reaction products. (A) p53Pro sequenced by the reverse primer. (B) p53Arg sequenced by the forward primer.

The distribution of p53 codon 72 genotypes in samples from 50 healthy blood donors from Beijing were as follows: Ten of 50 samples (20%) were p53Arg homozygotes, 14 of 50 samples (28%) were p53Pro homozygotes, and 26 of 50 samples (52%) were heterozygotes. The distribution of p53 codon 72 genotypes in samples from high-incidence and low-incidence populations for esophageal carcinoma as well as samples of esophageal, ovarian, and breast carcinoma is summarized in Table 1. The distribution of the p53 codon 72 polymorphism (C/G) showed no significant differences between the Beijing population and the Anyang population (chi-square test, 0.685; degrees of freedom [df] = 2; P = 0.710). However, there were significant differences in the frequency of the Arg genotype between HPV positive and HPV negative carcinoma samples and between HPV positive carcinoma samples and the control samples. The p53Arg homozygotes were significantly higher in HPV positive carcinoma samples. The p53 codon 72 genotypes in HPV positive samples of different histologic grades of esophageal mucosa and in HPV positive carcinoma samples are shown in Table 2. The distribution of the p53 codon 72 polymorphism (C/G) among different histologic grades of HPV positive esophageal epithelium showed no significant differences (chi-square test, 0.041; df = 2; P = 0.980). Similar results were found in HPV negative samples. However, the frequency of p53Arg homozygotes in HPV positive carcinoma samples was significantly higher compared with HPV positive samples from normal esophageal epithelium (P = 0.003, P = 0.035, and P = 0.049, respectively).

Table 1. Distribution of Three Genotypes of the p53 Gene at Codon 72 in Esophageal Carcinoma among High-Incidence and Low-Incidence Populations and Patients with Squamous Cell Carcinoma of the Esophagus, Ovarian Carcinoma and Breast Carcinoma
GroupNo.No. with genotype (%)Chi-squareP value95%CI
Arg/ArgArg/ProPro/Pro
  1. 95%CI: 95% confidence interval; HPV: human papilloma virus.

High-incidence population4810 (21)24 (50)14 (29)0.10–0.34
Low-incidence population339 (27)17 (52)7 (21)0.15–0.49
Beijing population5010 (20)26 (52)14 (28)0.6850.7100.10–0.34
Esophageal carcinoma6227 (43)21 (34)14 (23)6.9380.008
 HPV positive3921 (53)10 (26)8 (21)11.0570.001
 HPV negative236 (26)11 (48)6 (26)0.3410.559
Ovarian carcinoma3914 (36)20 (51)5 (13)2.8120.094
 HPV positive2611 (42)12 (46)3 (12)4.2570.039
 HPV negative133 (23)8 (62)2 (15)0.0601.000
Breast carcinoma2811 (39)10 (36)7 (25)3.3930.065
 HPV positive199 (47)6 (32)4 (21)5.1680.023
 HPV negative92 (22)5 (56)2 (22)0.0231.000
Table 2. p53 Codon 72 Genotypes in Human Papillomavirus Positive Samples of Different Pathologic Types of Esophageal Mucosa and Ovarian and Breast Carcinoma
Pathologic typesNo.No. with genotype (%)Chi-squareP value95%CI
Arg/ArgArg/ProPro/Pro
  1. 95%CI: 95% confidence interval.

Normal mucosa174 (24)9 (52)4 (24)0.0410.9800.06–0.44
Mild dysplasia235 (22)11 (48)7 (30)0.09–0.49
Severe dysplasia82 (25)4 (50)2 (25)0.03–0.56
Total4811 (23)24 (50)13 (27)
Esophageal carcinoma3921 (53)10 (26)8 (21)8.8520.003
Ovarian carcinoma2611 (42)12 (46)3 (12)2.0050.035
Breast carcinoma199 (47)6 (32)4 (21)3.8870.049

DISCUSSION

The p53 gene is one of the most intensely studied human genes because of its role as a tumor suppressor gene.17 Mutations in p53 are found in over 50% of all human malignancies, indicating that there is a powerful selection for loss of p53 activity during tumor development.17, 18 Normally, p53 arrests the cell cycle at the G1/S check point when it detects genotoxic damage of the DNA strand integrity, thus allowing DNA repair or apoptosis and avoiding replication of a damaged template. Mutation of p53 results in the loss of tumor suppressor function, which has been associated with cell carcinogenesis.17 Wild type p53 is polymorphic at codon 72 in human populations, and it has been found that this polymorphism is a high-risk factor for HPV-associated cervical carcinoma.7 In the current study, we used an allele specific PCR method to analyze the p53 codon 72 (C/G) polymorphism in HPV-associated malignancies among the Chinese population.

Table 1 shows that there were no significant differences in the distribution of p53 polymorphism between the Beijing population and the Anyang population (chi-square test, 0.685; df = 2; P = 0.710). Because our previous studies indicated a high HPV prevalence rate in Anyang among the high-incidence population for esophageal carcinoma (HPV-16, 72%; HPV-18, 17%),14 the results suggest that p53 codon 72 polymorphism has no effect on HPV infection. However, the frequency of p53Arg homozygotes in samples of esophageal, ovarian, and breast carcinoma were significantly higher in HPV-positive samples (53%, 42%, and 47%, respectively), whereas, in HPV negative samples, the p53Arg homozygotes comprised 26%, 23% and 22%, respectively, similar the control group (P = 0.559, P = 1.000, and P = 1.000, respectively). Our results strongly suggest that the p53Arg homozygote genotype is one of the high-risk genetic factors for HPV-associated malignancies among the Chinese population. It has been reported that these two polymorphic variants of wild type p53 differ biochemically and biologically.19 p53Arg is significantly more susceptible compared with p53Pro to the degradation induced by high-risk HPV E6 protein and the resulting loss of function. Normally, p53 acts as a genome guard. Loss of p53 function may increase the probability of carcinogenesis in HPV-infected cells. Accordingly, individuals who are homozygous for p53Arg are more susceptible to carcinogenesis after HPV infection. Our data also may explain the controversial reports of the correlation of p53 codon 72 polymorphism with a susceptibility to carcinoma. Most of the previous studies did not separate the HPV positive and negative samples when p53 codon 72 polymorphism was checked and analyzed.

In addition, our previous studies showed that HPV was present in different histologic grades of esophageal epithelium from normal mucosa, to mild dysplasia, to severe dysplasia; and, with the progression of histologic grade, the HPV infection rate and viral oncogene expression increased.14 According to Table 2, the distribution of p53 codon 72 polymorphism (C/G) among different histologic grades of HPV positive esophageal epithelium showed no significant differences (chi-square test, 0.041; df = 2; P = 0.980). Similar results were found in HPV negative samples. These results suggest that p53 polymorphism may not be related to an increased HPV infection rate or to the progression of cytopathologic grade in esophageal epithelium, implying that p53 polymorphism may have less importance in the susceptibility to carcinoma until the last step in carcinogenesis. In other words, the p53 codon 72 polymorphism (C/G) may be a factor for susceptibility only in the very late stage of carcinogenesis.

In summary, the current study suggests that the p53 codon 72 Arg homozygous genotype is one of the high-risk genetic factors for HPV-associated malignancies among the Chinese population. However, it is accepted widely that many environmental factors and genes are involved in the multiple steps of carcinogenesis, and the p53 gene may be only one of them. Thus, it will be important in future studies to investigate the germline polymorphism of other related genes, for example, the genes in the p53 pathway.

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