Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Abstract

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.

Figure 1. G2 checkpoint defect induced by LMP1.

A and B, Relative mitotic index at different time points after γ-ray irradiation (relative percentage of mitotic index as compared with unirradiated cells). Error bars represent standard deviations. Arrows indicated the relative mitotic indices in LMP1-expressing cells significantly higher (P<0.05) than empty vector-infected control cells. C, Examples of G2 cells identified by pan-nuclear CENP-F staining. DNA was stained by DAPI. D and E, Quantification of percentage of G2 cells with or without γ-ray irradiation. Error bars represent standard deviations. Stars indicated significant differences (P<0.05) between the indicated datum pairs.

Figure 2. Frequencies of chromatid breaks in LMP1-expressing and control cells.

A, Examples of chromatid breaks 3 h after 0.5 Gy γ-irradiation. The nascent chromatid breaks (as indicated by arrows) were identified by the chromatid gap larger than the width of the chromatid and the lack of telomere signals. The short arrows indicate the subtle terminal chromatid breaks. Telomeres were detected by red signals and DNA was stained blue by DAPI. B and C, Frequencies of chromatid breaks before or after γ-irradiation. Error bars represent standard deviations. For all time points analyzed, the frequencies of chromatid breaks in LMP1-expressing cells were significantly higher (P<0.05) than empty vector-infected cells.

Figure 3. Western Blotting analysis in LMP1-expressing and control cells.

Protein expression analysis by Western Blotting of various proteins related to Chk1 activation in cells without γ-irradiation or at different time points after 0.5 Gy γ-irradiation. Actin shows protein loading controls.

Figure 4. G2 checkpoint improvement in HONE1-LMP1 cells with ectopic Chk1 overexpression.

A, Western Blotting analysis of Chk1, p-Chk1(S345), p-Cdc2(Y15) and Cdc2. Actin shows protein loading controls. B, Relative mitotic index at different time points after γ-ray irradiation. Error bars represent standard deviations. Arrows indicated the relative mitotic indices in HONE1-LMP1 cells transfected with control plasmids (pEGFP) significantly higher (P<0.05) than HONE1-LMP1 cells transfected with Chk1-expressing plasmids (pEGFP-Chk1). C, Frequencies of chromatid breaks before or after γ-irradiation. Error bars represent standard deviations. Stars indicated significant differences (P<0.05) between the indicated datum pairs.