VIRAL E6-E7 TRANSCRIPTION IN THE BASAL LAYER OF ORGANOTYPIC CULTURES WITHOUT APPARENT P21CIP1 PROTEIN PRECEDES IMMORTALIZATION OF HUMAN-PAPILLOMAVIRUS TYPE-16-TRANSFECTED AND TYPE-18-TRANSFECTED HUMAN KERATINOCYTES

Organotypic cultures of human keratinocytes provide a useful model sys tem to study human papillomavirus (HPV)-host cell interactions. In thi s study, we analyzed organotypic cultures of two HPV type 16 (HPV16) ( FK16A and FK16B)- and two HPV18 (FK18A and FK18B)-transfected keratino cyte cell lines through the process of immortalization in vitro. For F K16A and FK18B cells, passages of both mortal cells in their extended life span and subsequent immortal stages were studied. Mortal cells of FK16A and FK18B showed a morphology reminiscent of mild to moderate d ysplasia, whereas in their immortal descendants, severely dysplastic f eatures were observed. Immortal FK18A cells were mildly to moderately dysplastic, while FK16B cells were severely dysplastic. The increasing degrees of dysplasia were associated with a decreasing expression of differentiation markers cytokeratin 10 and profilaggrin. All raft cult ures expressed E6-E7 mRNAs in the basal layer, while the amount of vir al transcripts in the suprabasal cells was in general proportional to the degree of dysplasia. In all cases, E6-E7 transcription and dysplas tic features were highly correlated with cellular proliferation, as as sessed by Ki-67 (MIB-1) antigen expression. Moreover, high levels of E 6-E7 transcription and expression of p21cip1 protein in the basal laye r seemed to be mutually exclusive. We conclude that expression of E6-E 7 in the basal cells associated with increased proliferation in the ab sence of detectable p21cip1 protein is apparently necessary but not su fficient for immortalization, or for the loss of terminal differentiat ion, for which yet to be discovered additional events are required. Th e model system described in this study provides a valuable tool to ana lyze alterations in viral transcription regulation during HPV-mediated cell transformation.