J Korean Med Sci.  2005 Aug;20(4):636-642. 10.3346/jkms.2005.20.4.636.

Epstein-Barr Virus and p16INK4A Methylation in Squamous Cell Carcinoma and Precancerous Lesions of the Cervix Uteri

• Kim NR
• Lin Z
• Kim KR
• Cho HY
• Kim I

• KMID: 1712746
• DOI: http://doi.org/10.3346/jkms.2005.20.4.636

Abstract

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.

Keyword

Protein p16; Methylation; Herpesvirus 4, Human; Epstein-Barr Virus Infections; Cervix Neoplasms; Carcinogenesis

MeSH Terms

Carcinoma, Squamous Cell/genetics/*pathology/virology
Comparative Study
Cyclin-Dependent Kinase Inhibitor p16/analysis/*genetics
*DNA Methylation
DNA, Viral/genetics/isolation & purification
Epstein-Barr Virus Infections/genetics/*pathology/virology
Epstein-Barr Virus Nuclear Antigens/genetics
Female
Herpesvirus 4, Human/genetics
Humans
Immunohistochemistry
In Situ Hybridization
Polymerase Chain Reaction
Precancerous Conditions/genetics/*pathology/virology
RNA, Viral/genetics
Research Support, Non-U.S. Gov't
Uterine Cervical Neoplasms/genetics/*pathology/virology

Figure

• 

  Fig. 1 Methylation status of the p16 gene detected by methylation-specific PCR. Lane 1-3, CIN; lane 4-6, cervical carcinoma; lane 7, normal cervical tissue; M, Molecular weight marker; lanes m, reactions using p16-M primers specific for the methylated CpG sites. lanes u, reactions using p16-U primers specific for the unmethylated CpG sites.

• 

  Fig. 2 Immunohistochemistry for p16. Focal nuclear staining in the basal cells of the non-neoplastic cervical tissue (A, p16 immunostain, ×200), CIN I (B, p16 immunostain, ×200), CIN III (C, p16 immunostain, ×200) and intense nuclear staining in invasive squamous cell carcinoma (D, p16 immunostain, ×400).

• 

  Fig. 3 Analysis of EBV using EBNA-1 primer. Lane 1-2, Normal cervical tissue; lane 3-6, Squamous cell carcinoma; lane 7-9, CIN; lane N, Negative control; lane C, Positive control; M, molecular weight marker.

• 

  Fig. 4 (A) EBER-1 signals detected by in situ hybridization (×200). No signals are detected in squamous cell carcinoma. (B) In CIN I, some of the dysplastic cells show nuclear staining (×200). (C) In CIN III, dysplastic cells show nuclear staining (×400). (D) In squamous cell carcinoma, tumor cells and some intervening lymphocytes show nuclear staining for EBER-1 (×400).

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