Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells.
Integration of episomal human papillomavirus (HPV) DNA in infected cervical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied immortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ectocervical keratinocytes was compared with that in the same cells after crisis and establishment of immortalization. Southern blots revealed, and two-dimensional gel analysis confirmed, that the precrisis culture contained more than 100 copies/cell of episomal HPV-18 DNA and no detectable integrated viral DNA. In contrast, the postcrisis cells contained a low copy number of only integrated viral genome. The Northern blot patterns of E6-E7 and E2/E4 RNA expression were also different. Analysis of RNA by RT-PCR indicated that neither culture expressed the unspliced HPV-18 E6 oncogene present in tumor cell lines and that the precrisis, but not postcrisis, culture expressed the full-length E2 repressor. The two cultures displayed a similar keratinocyte morphology in vitro and a similar low grade dysplasia in vivo and both were non-tumorigenic. These results suggest that, although insufficient for complete malignant conversion, viral DNA integration during crisis is associated with the establishment of an immortalized phenotype in which HPV-18 DNA is integrated and HPV-18 RNA expression is altered.
D000328:Adult; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002584:Cervix Uteri / Q000166:cytology / Q000821:virology*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000821:virology; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics*; D012326:RNA Splicing; D012367:RNA, Viral / Q000032:analysis; D016662:Virus Integration
D004279:DNA, Viral; D012367:RNA, Viral