Abstract
BackgroundClaspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint. Claspin expression is significantly high in several human solid tumors. Furthermore, high levels of claspin have been found in cervical cancer cell lines. Nevertheless, no data are available regarding claspin expression in cervical tissues.MethodsIn order to investigate whether claspin immunoreactivity is related to the lesion severity and High-Risk (HR) HPV infection, we analyzed claspin expression by immunohistochemistry in a series of cervical biopsies which represent the steps occurring during cervical carcinogenesis (normal tissues, Cervical Intraepithelial Neoplasias 1, 2 and 3, Squamous Cell Carcinomas). All patients also had a cervico-vaginal sample for HPV testing, collected immediately before the colposcopy-guided biopsy. The HR-HPV DNA detection was performed by the HR-HPV Hybrid Capture 2 test. HPV genotyping was performed using the Linear Array HPV Genotyping Test.ResultsOur results evidenced a constant and significant increase of the rate of claspin positivity from the normal tissues to carcinomas (pχ2trend < 0.0001). In fact, the normal tissues displayed either no or faint claspin immunoreactivity, whereas a moderate/high positivity was observed in 16% of the CIN1, 76% of the CIN2, 87.5% of the CIN3 and 93.3% of the cancers. Moreover, we found a statistically significant correlation between claspin expression and HR-HPV infection (pχ2 < 0.0001), irrespective of the genotype. Finally, we demonstrated the feasibility of claspin immunostaining in cervical cytology.ConclusionsOur findings indicate that in vivo claspin expression is significantly related to HR-HPV infection and lesion grade both in histological and cytological samples. Therefore, the analysis of claspin expression could be clinically relevant in the diagnosis of HPV-related cervical lesions, in particular when applied to cervico-vaginal cytology. Moreover, giving information on the proliferation rate of each lesion, claspin immunostaining may contribute to the evaluation of progression risk, thus being helpful in patient management. Nevertheless, only large prospective studies may clarify the true clinical usefulness of claspin expression in distinguishing lesions with different progression potential.
Highlights
Claspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint
In vitro claspin expression In order to investigate the correlation between claspin expression and uterine cervix transformation, we examined the expression of claspin by western blotting both in normal human keratinocytes (NHEK) and in a series of cell lines that resembled cervical malignancy at different stages of tumorigenesis (HK-168, SiHa, CaSki, HeLa)
We found no claspin expression in the normal cell line and in the HK-168 cell line which has a basal/ parabasal keratinocyte phenotype resembling the CIN2 stage [14], while its expression was evident in SiHa, CaSki and HeLa cervical carcinoma cell lines (Figure 1)
Summary
Claspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint. Claspin expression is significantly high in several human solid tumors. High levels of claspin have been found in cervical cancer cell lines. No data are available regarding claspin expression in cervical tissues. Claspin is a nuclear protein involved in both DNA replication and damage response and is a key mediator for the S-phase checkpoint. Inhibition of claspin, as well as ATR and Chk, greatly various steps of cellular atypia and its cause, i.e. the High Risk Human Papillomavirus (HR-HPV) infection, is well established. Recent in vitro data showed high claspin baseline level in cervical cancer cell lines. The cell lines expressing the HPV-E7 oncoprotein showed an increased claspin turnover [8]
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