The p53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins with C-terminal BRCA1 C-terminus domains.^3,4 53BP1 is a nuclear protein that rapidly localizes at the sites of DNA double strand breaks (DSBs) and activates p53 along with other kinases.^5–10 Activated p53 plays critical roles in the DDR, such as cell cycle arrest, DNA repair, and apoptosis.^11,12 It has been well documented in vitro with immunofluorescence that 53BP1 exhibits diffuse nuclear staining in untreated primary cells, whereas, after exposure to radiation, 53BP1 localizes at the sites of DSBs and forms discrete nuclear foci (NF).^5,6,13,14 We have recently demonstrated that, with an immunofluorescence method, 53BP1 NF may serve as a valuable molecular marker of GIN during carcinogenesis.^15,16 GIN seems to be induced at the precancerous stage during thyroid and skin carcinogenesis, as follicular adenoma and actinic keratosis show occasional 53BP1 NF.^15,16 Given that one manifestation of GIN is the induction of endogenous DDR,¹⁷ we propose that immunofluorescence analysis of 53BP1 expression can be a useful tool with which to estimate the level of GIN as well as the malignant potential of human tumours.

Uterine cervical cancers are believed to develop through a multistep process. Furthermore, it is well established that persistent infections with high-risk human papillomavirus (HR-HPV) represent a necessary cause of high-grade premalignant lesions and subsequent invasive cancer of the uterine cervix. The HR-HPV viral oncogenes, E6 and E7, have been shown to be the main contributors to the development of human papillomavirus (HPV)-induced cervical cancer, and increased expression resulting from integration of the viral DNA into the host genome has been detected in invasive cancers and a subset of high-grade lesions.¹⁸ It has been shown that E6 and E7 together cause polyploidy soon after they are introduced into cells, so GIN is thought to play an essential role in the cellular transformation of cervical epithelium during carcinogenesis. The most manifest function of the E6 protein is to promote the degradation of p53 through its interaction with a cellular protein, E6-associated protein, an E3 ubiquitin ligase.¹⁹ E7 is known to bind to the retinoblastoma tumour suppressor gene product, retinoblastoma protein (pRb). Phosphorylation of pRb by G₁ cyclin-dependent kinases releases E2F, leading to cell cycle progression into the S phase. E7 is able to bind unphosphorylated pRb, and this may induce cells to prematurely enter the S phase by disrupting pRb–E2F complexes. One cyclin-dependent kinase inhibitor, p16^INK4a, which prevents the phosphorylation of pRb family members, is overexpressed when pRb is inactivated by E7.²⁰ Normally, overexpression of p16^INK4a results in cell cycle arrest, but with E7 expression, this is overcome. Thus, overexpression of p16^INK4a has been suggested as a useful biomarker for evaluating HPV pathogenic activity in cervical lesions.

The value of p16^INK4a as a surrogate marker of HR-HPV and CIN has been well established in recent years, with studies showing both increased immunoexpression of p16^INK4a in dysplastic cervical epithelium and a positive correlation with HR-HPV infection and the degree of CIN.^24–30 Cervical SCC develops through a multistep process that involves replication stress. An important occurrence in cervical carcinogenesis is deregulated expression of the HR-HPV oncogenes E6 and E7. Several risk factors for cervical neoplastic progression are likely to contribute to viral oncogene deregulation, particularly integration of HR-HPV into the host genome.^31,32 We found a significant association between the type of HR-HPV ISH signal and type of 53BP1 expression in the cervical epithelium. The distribution of 53BP1 NF was similar to that of the punctate type of ISH signals in both CIN and SCC, suggesting that viral integration could induce endogenous DSBs through the induction of GIN in the host genome. Furthermore, in cervical lesions, the distribution of 53BP1 NF was identical to that of p16^INK4a overexpression; this represents a byproduct of viral infection, and suggests an association between the presence of GIN and replication stress that allows enhancement of cell proliferation and avoidance of cell death. As co-localization of 53BP1 NF and the punctate type of HR-HPV ISH signal was rare, most viral integrated sites might have been bypassed by the host's DDR machinery, and integrants may have a growth advantage over others in a mixed population of cervical cells.

This study also demonstrated little co-localization of the DDR type of 53BP1 expression and Ki67 nuclear staining as a marker for cycling cells in precancerous lesions. An intact DDR pathway is activated, leading to cell cycle arrest by p53 activity, so precancerous/dysplastic cells may still preserve DDR function, at least partially, and this may induce sporadic regression of dysplasia. In contrast, cancer cells showing 53BP1 NF were frequently co-localized with Ki67 staining, suggesting a disruption of the DDR pathway that subsequently leads to an irreversible malignant transformation. Taken together, these findings indicate that GIN may have already occurred at the precancerous stage during cervical carcinogenesis, and that increasing GIN based on a disrupted DDR may allow further accumulation of other genomic alterations, causing progression to invasive cancer through acceleration of cell growth/replication stress. Further research is needed on the molecular mechanism of the uncoupling of cell cycle progression in the face of increasing severity of DNA damage at the advanced phase of carcinogenesis.