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 cer vical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied i mmortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ect ocervical 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 cultur e contained more than 100 copies/cell of episomal HPV-18 DNA and no de tectable integrated viral DNA. In contrast, the postcrisis cells conta ined a low copy number of only integrated viral genome. The Northern b lot patterns of E6-E7 and E2/E4 RNA expression were also different. An alysis of RNA by RT-PCR indicated that neither culture expressed the u nspliced HPV-18 E6 oncogene present in tumor cell lines and that the p recrisis, but not postcrisis, culture expressed the full-length E2 rep ressor. The two cultures displayed a similar keratinocyte morphology i n vitro and a similar low grade dysplasia in vivo and both were non-tu morigenic. These results suggest that, although insufficient for compl ete malignant conversion, viral DNA integration during crisis is assoc iated with the establishment of an immortalized phenotype in which HPV -18 DNA is integrated and HPV-18 RNA expression is altered.