Codon 72 polymorphism of p53 as a risk factor for patients with human papillomavirus-associated squamous intraepithelial lesions and invasive cancer of the uterine cervix

Tsuyoshi Yamashita2, Yuji Yaginuma, Yuji Saitoh, Kiitirou Kawai, Toshiyuki Kurakane1, Hiroaki Hayashi and Mutsuo Ishikawa

Department of Obstetrics and Gynecology, Asahikawa Medical College, Nishikagura 4-5-3-11, Asahikawa 078-8510, Japan


    Abstract
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 Abstract
 Introduction
 References
 
Squamous intraepithelial lesions (SIL) and invasive cancer of the uterine cervix are thought to be a series of lesions derived from normal cervical squamous tissue. Infection by high risk human papillomavirus (HPV) and integration of viral DNA may initially lead normal cervical cells to become pre-malignant cells in SIL and result in cervical malignancies later on. High risk HPVs, including types 16 and 18, produce a viral protein, E6, which is required for viral replication in host cells. The E6 protein is able to bind to host p53 causing inactivation of its function through the mechanism of ubiquitin-dependent degradation. It has recently been reported that the extent of p53 dysfunction caused by HPVs depends on the status of a polymorphism at codon 72 of p53, Pro or Arg. In that study, it was demonstrated that a patient homozygous for the Arg allele had about a seven times higher risk of developing cervical cancer than a patient homozygous for Pro. In an attempt to confirm this result and elucidate whether this allelic deviation of the Arg genotype seen in invasive cervical cancer occurs in the pre-malignant lesion SIL, we analyzed 219 SIL and 101 invasive cancer samples from Japanese patients using a PCR-based assay. Samples from 88 SIL and 76 invasive cancers were identified as HPV-infected samples and used for further analyses. In these, the frequencies of Arg homozygotes were 31.8, 33.0 and 36.8% in controls, SIL and invasive cancer, respectively. The distributions of the different alleles of codon 72 (Pro/Pro, Pro/Arg and Arg/Arg) did not show significant differences between either control and SIL groups or control and invasive cancer groups. Also, no difference in the frequency of Arg/Arg genotype was detected even between the control and HSIL groups or control and invasive cancer infected with high risk HPVs groups. In conclusion, there was no obvious relationship between the Arg genotype at codon 72 of p53 and predisposition to HPV-associated cervical neoplasia.

Abbreviations: HPV, human papillomavirus; SIL, squamous intraepithelial lesions.


    Introduction
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 Abstract
 Introduction
 References
 
Human papillomavirus (HPV) is one of the important candidates for malignant conversion of uterine cervical tissue (1). A wide variety of HPVs have been found in tissue-derived squamous intraepithelial lesions (SIL) and invasive cancers of the uterine cervix (2). In particular, several HPVs, including types 16 and 18, termed high risk types, are frequently detected in high SIL and/or more advanced lesions (2,3). Host cells with integrated viral DNA fragments from high risk HPVs become a source of malignant cells seen in advanced stages of uterine cervical cancer (4,5), although other factors may be required for complete malignant transformation to invasive cancer (6,7). Interestingly, a recent study by Storey et al. appears to give us a new insight into the mechanism of cervical carcinogenesis, demonstrating that individuals with Arg alleles at codon 72 of p53 are predisposed to HPV-associated cervical cancer compared with those with Pro alleles (8). This suggests that not only the presence of HPV infection but a structural difference in p53, at least in part, may be an important factor to consider for cervical carcinogenesis. This also implies that genotyping of Pro/Arg alleles in patients with SIL could allow us to select patients at high risk for invasive cervical cancer because SIL is believed to be a pre-malignant lesion leading to invasive cancer (1). In this study, we investigated 252 samples of normal individuals and 320 samples from SIL and invasive cervical cancer using a PCR-based assay to confirm the previously reported result and to find out whether this allelic deviation of the Arg genotype seen in invasive cervical cancer occurs in SIL.

As sources of DNA from SIL and invasive cancer, 320 cytological and paraffin-embedded tissue samples from Japanese women who had undergone cytological examination of the uterine cervix or primary surgery for uterine cancer were used. Diagnosis of all samples used in this study, including cytological samples, was confirmed by histopathological examination. As controls, 252 cytological samples diagnosed as `within the normal limit' by The Bethesda System (TBS) (9) were used. These samples were retrieved from the Asahikawa Detection Center of Hokkaido Cancer Society, Asahikawa Medical College and its related hospitals. Genomic DNA from cytological samples was extracted with Isogen/Isogen-LSTM (Nippon Gene, Tokyo, Japan) or DNA extractor WB kitTM (Wako Pure Chemical Industries, Osaka, Japan) using the manufacturer's protocols. For paraffin-embedded tissues, three 10 µm sections were cut from a tissue block and DNA was extracted with DEXPATTM (Takara, Tokyo, Japan) as described by the manufacturer. Extracted DNA solution was again purified with phenol/chloroform followed by ethanol precipitation. DNA from all samples was finally dissolved into distilled water in volumes of 30–300 µl.

To detect HPV DNA, two different consensus primer sets, C and D, were used for PCR amplification (Figure 1Go). These primer sets are at least able to amplify sequences of HPV types 6, 11, 16, 18, 31, 33, 39, 45, 51 and 56 (10,11). Each 25 µl of PCR mixture contained 2 µl of sample DNA solution containing 25–250 ng of genomic DNA fragment, 0.35 µM primers, 2.5 mM MgCl2, 0.25 mM dNTP, 2.5 µl of supplied PCR buffer and 0.5 U of Taq polymerase (Boehringer Mannheim). PCR was carried out as previously described (10,11) and the product was electrophoresed on 2% agarose gels stained with ethidium bromide and viewed under UV light. Samples identified as HPV-positive with these consensus primers were then HPV genotyped with type 16 and 18 specific primers (12) (Figure 1Go). For analysis of alleles with Arg or Pro at codon 72, modified PCR–RFLP (non-radioactive) was applied (Figure 2a and bGo). A reverse primer B which contains one nucleotide substitution (Figure 1Go) enabled the creation of two different restriction sites, KspI(SacII) and SmaI, in the product (Figure 2aGo). These two sites allowed us to distinguish the sequences CCC for Pro and CGC for Arg at codon 72. Application of two different enzymes to the same region can reduce misjudgement of partial digestion of the PCR product (Figure 2bGo). PCR was performed with the same mixture as described above except for primers using 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The product was purified with phenol/chloroform followed by ethanol precipitation, then digested with either KspI(SacII) (37°C) or SmaI (25°C) overnight. All of the digested products were electrophoresed through a 2% agarose gel stained with ethidium bromide and genotyped by their digestion patterns.



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Fig. 1. Partial p53 sequence showing a fragment generated by PCR and the primers for detection of p53 Pro/Arg alleles and HPVs. Sequences of primers situated in intron 3 and exon 4 are underlined. The sequence of intron 3 is shown in lower case letters and that of exon 4 in upper case. A polymorphic site of codon 72 (P) is indicated in bold with underlining. Primer B contains a nucleotide substitution (*) to create KspI(SacII) and SmaI restriction sites in the product. Primers to detect HPV sequences used in this study are shown at the bottom. Consensus primer set C was used to detect HPV types 6, 11, 16, 18, 31 and 33; consensus primer set D for types 18, 39, 45, 51 and 56.

 


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Fig. 2. Schematic representation of the sequence of the PCR product containing a Pro or Arg allele. (a) Two polymorphic forms amplified with reverse primer B containing a nucleotide substitution. SmaI is only able to cut the product (162 bp) with the Pro allele into a 135 and a 27 bp fragment. KspI(SacII) can cut the product with the Arg allele into fragments of the same size. (b) PCR products digested by restriction enzymes. Ds, digestion with SmaI; Dk, digestion with KspI(SacII). Sample A shows complete digestion in Dk and no digestion in Ds, indicating the homozygous Arg allele alone in the product, and sample B contains both the Pro and Arg alleles. Sample C shows partial digestion in Ds due to the large amount of the product, but C with Dk displays no digestion, indicating a homozygous Pro allele in this product.

 
It has been reported that the frequency of the p53 Pro/Arg polymorphism varies with ethnic group (13). Thus we first examined the frequency of the Pro/Arg polymorphism in samples from a control group. As shown in Table IGo, the frequencies of Pro/Pro, Pro/Arg and Arg/Arg were 14.3, 54.0 and 31.7%, respectively. The distributions of genotype in the control group was similar to those previously described (14,15).


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Table I. Distribution of p53 codon 72 polymorphic forms
 
In invasive cancer, HPV DNA was detected in 76 out of 101 examined samples. In the HPV-positive cases, the allelic frequencies of Pro/Pro, Pro/Arg and Arg/Arg were 5.3, 57.9 and 36.8%, respectively (Table IGo). HPV 16 and 18 were detected in 60 samples out of 76 samples and frequencies of the genotypes at codon 72 were 5.0, 58.3 and 36.7%, respectively. Like other reports (1519), our results show no significant difference in the frequency of the Arg/Arg genotype between controls and HPV-positive patients (odds ratio 0.78, 95% confidence interval 0.56–1.33, P = 0.41). Moreover, no significant difference between controls and patients with high risk HPV (16 and 18)-associated cancer was found (odds ratio 0.80, 95% confidence interval 0.54–1.39, P = 0.47).

In SIL, samples were analyzed by the same method as that for invasive cancer. HPV-positive SIL samples were detected in 92 out of 214 samples. Frequencies of the three genotypes in these HPV-positive SIL samples were 15.9 (Pro/Pro), 51.1 (Pro/Arg) and 33.0% (Arg/Arg), respectively (Table IGo). Little difference in the allele distribution was observed even between the control and high SIL groups. Statistical analyses showed no significant difference in the frequency of the Arg/Arg genotype between the control and SIL groups (odds ratio 0.95, 95% confidence interval 0.63–1.47, P = 0.83) and the control and high SIL groups (odds ratio 0.98, 95% confidence interval 0.59–1.65, P = 0.95).

To date, no report has supported the result that the Arg/Arg genotype may be strongly involved in the incidence of HPV-associated cervical malignancies; there may be some reason why this result cannot be reproduced in other investigations (16,17). However, in our current study the results again do not indicate that allele distribution at codon 72 of p53 shows any significant differences among the control, SIL and invasive cancer groups. This suggests that a structural difference at codon 72 of p53 may not influence the process of cervical carcinogenesis or may not be a dominant factor for predisposition to invasive cervical cancer.


    Acknowledgments
 
We thank K.Kirio for collecting samples. This study was supported by a grant-in-aid for General Scientific Research no. 09771251 from the Ministry of Education, Science, Sports and Culture, Japan.


    Notes
 
1 Present address: Department of Cytology, Asahikawa Detection Center of Hokkaido Cancer Society, Suehiro-higashi 2-6, Asahikawa 071-8122, Japan Back

2 To whom correspondence should be addressed Email: tyamashi{at}asahikawa-med.ac.jp Back


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Received January 4, 1999; revised April 29, 1999; accepted May 12, 1999.