Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.

SummaryThe need for antiviral therapies for papillomavirus infections is well recognized but the difficulties of reproducing the infectious cycle of papillomaviruses in vitro has hindered our understanding of virus-cell interactions and the regulation of viral gene expression during permissive growth. Recent advances in understanding the temporal expression and function of papillomavirus proteins. has enabled consideration of a targeted approach to papillomavirus chemotherapy and in particular the inhibition of viral replication by targeting the El and E2 proteins. There are in vitro culture systems available for the screening of new chemotherapeutic agents, since significant advances have been made with culture systems which promote epithelial differentiation in vitro. However, to date, there are no published data which show that virions generated in vitro can infect keratinocytes and initiate another round of replication in vitro. In vivo animal models are therefore necessary to assess the efficacy of antivirals in preventing and treating viral infection, particularly for the low-risk genital viruses which are on the whole refractory to culture in vitro. Although papillomaviruses affect a wide variety of hosts in a species-specific manner, the animals most useful for modelling papillomavirus infections include the rabbit, ox, mouse, dog, horse, primate and sheep. The ideal animal model should be widely available, easy to house and handle, be large enough to allow for adequate tissue sampling, develop lesions on anatomical sites comparable with those in human diseases and these lesions should be readily accessible for monitoring and ideally should yield large amounts of infectious virus particles for use in both in vivo and in vitro studies. The relative merits of the various papillomavirus animal models available in relation to these criteria are discussed.

We have compared the responses of normal human cervical keratinocytes (HCE) to diethylstilboestrol (DES), and the promoting agents, phorbol-12-myristate-13-acetate (PMA) and mezerein using the loss of cloning efficiency as a measure of terminal differentiation in vitro. Dose-response studies showed that normal HCE are growth inhibited by chronic exposure to DES at concentrations greater than or equal to 2.5 X 10(-5) M, to PMA at concentrations greater than 10(-8) M and mezerein at concentrations greater than 10(-9) M. Compared to acetone controls, promoter or DES-treated cells exhibited a 10- to 12-fold increase in cornified-envelope formation. Normal HCE exhibit a heterogeneous response to PMA in that 85-90% of colony-forming cells lose their colony-forming ability after a 24-h exposure to 10(-6) M PMA. The PMA-resistant subpopulation, PMAR, remains constant and is not reduced even after 96 h chronic exposure to PMA. In contrast, the colony-forming ability of normal HCE is almost totally suppressed after 24 h exposure to 10(-6) M mezerein. After 24 h incubation with 5 X 10(-5) M DES, 20% of normal HCE are capable of colony formation but this resistant fraction is eliminated after 96 h chronic exposure. Cornified-envelope formation was negligible in malignant cervical keratinocytes grown in the presence of DES or promotors and these cells were characterised by a very large PMAR fraction - 85 - 90% of cells retained colony-forming ability after exposure to 10(-6) M PMA for 24 h. Furthermore, 90-100% of malignant cervical keratinocytes retained their colony-forming capacity after exposure to 10(-6) M mezerein. However, colony-forming ability declined steadily in the presence of 5 X 10(-5) M DES and after 96 h only a tiny fraction, 1% of malignant cervical keratinocytes could form colonies on replating. The mechanisms by which DES inhibits growth and induces cornified-envelope formation in HCE would appear to be distinct from those activated by PMA and mezerein.

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