The expression of p63 is associated with the differential stage in nasopharyngeal carcinoma and EBV infection

Abstract

Background: Nasopharyngeal carcinoma (NPC) is common among Southern Chinese and the main histology is the undifferentiated carcinoma associated with Epstein-Barr virus (EBV) infection. p63 is a recently proved member of the p53 family based on the structural similarity to p53, but its function in NPC is still unknown. This study was aimed to investigate the association between p63 and NPC.

Results: p63 was expressed in 100% (202/202) of nasopharyngeal carcinoma (NPC) tissues but not in 29 nasopharynx inflammation and 17 non-cancerous nasopharyngeal epidermises on a tissue microarray by immunohistostaining. Further investigation suggested that the p63 expression was associated with the differential stage of NPC: p63 strong staining in Keratinizing squamous cell carcinoma, differentiated non-keratinizing NPC and undifferentiated non-keratinizing NPC presented the percentage of 5/8 (62.5%), 43/48 (92.5%) and 50/50 (100%), respectively. A significant difference (p = 0.001) existed between the keratinizing and non-keratinizing groups. No pathogenic mutations were detected in p63 gene in 12 primary NPC tissues and matched peripheral blood lymphocytes (PBL). Half-life measurement study revealed distinct stability of p63 protein in the different cell lines, especially between the carcinoma cell lines with EBV infection and the non-cancerous cell lines. The results of immunoprecipitation suggested a direct interaction between Epstein-Barr virus nuclear antigen 5 (EBNA-5) and p63 protein in NPC, and this binding would increase the stability of p63.

Conclusion: Our data suggested p63 might be used as an adjunct diagnostic marker of NPC and contributed a new way to understand the contribution of the EBV in the pathogenesis of NPC.

Figure 1

Immunohistochemical demonstration of p63 protein expression. Strong positive staining of p63 protein in NPC(A) and the basal layer cells with proliferate potential(B); negative staining of p63 protein in nasopharynx inflammation (C) and non-cancerous nasopharyngeal epidermises(D). (All the photomicrographs were taken in high-powered, ×400).

Figure 2

Immunohistochemical demonstration of p63 protein expression in different histology NPC. Strong nuclear staining of p63 in keratinizing NPC (A) differentiated non-keratinizing NPC (B) and undifferentiated non-keratinizing NPC (C)(All the photomicrographs were taken in high-powered, ×400). The staining intensity is associated with the differential stage of NPC.

Figure 3

Stability of p63 with CHX treatment. CHX was added at 100 ug/ml and total protein was extracted as indicated. Western blots were performed with p63 and β-actin in (A) B95-8 cells, (B) C666 cells, (C) CNE2 cells, (D) CNE1 cells, (E) NP69 cells, (F) 293 cells.

Figure 4

Half-life of p63 in the different cell lines. The half life of p63 was 38.64h, 36.42h, 27.47h, 19.74h, 9.76h, 6.93h accordingly to B95-8cells, C666 cells, CNE2 cells, CNE1 cells, NP69 cells and 293 cells respectively.

Figure 5

Co-immunoprecipitation of p63 and EBNA-5. (A) Cell extract were precipitated with the JF186 and analysed by Western blot with p63. (B) Cell extract were precipitated with the p63 and analysed by Western blot with JF186.