Papillomavirus-Transformed Keratinocytes in Monolayer and Organotypic Cultures by Interferon-y and Tumor Necrosis Factor-a

The growth response ofnormal and humanpapilomavirus (HPV)-transformed cervical keratinocytes to interferon-y (IFN-,y) and tumor necrosis factor-a was investigated in monolayer and organotypic raft cultures. Theproliferation rates ofmonolayer cultures were assessed by [3HJTdR incorporation andfluorimetric DNA titration. The growth of keratinocytes in organotypic cultures was estimated by their ability to stratify on collagen rafts and by immunohistochemistry for Ki67 antigen expression. IFN-,y reduced the DNA synthesis ofnormal and HPV-transformed keratinocytes in monolayer cultures and exerted a marked growth inhibitory effect in organotypic raft cultures. In control raft cultures, normal keratinocytes produced an epithelial sheet of approximately 10 ceUs in tbickness that closely resembled normal cervical epithelium and was characterized by sparse Ki67 antigen-positive ceUls whereas HPV-transformed keratinocytes producedup to 15poorly differentiatedepithelial layers that were reminiscent of high grade cervical lesions seen in vivo and exhibited a fuU thickness Ki67 antigen expression. When normal and HPV-transformed keratinocytes were maintained in the presence of IFN-'y, the epithelial sheet was reduced to afew ceUls in thickness and the density ofKi67 antigen-positive ceUs was decreased. A morepronouncedgrowth inhibitory effect in monolayer and organotypic cultures was observed when IFN-,y was associated with tumor necrosisfactor-ax Tumor necrosisfactor-a alone reduced the DNA syntbesis of normal keratinocytes but was significantly less effective than IFN-,y to inhibit the growth ofHPV-transformed keratinocytes. These results suggest that similar responses in vivo to regulatory molecules may play a role in the development ofHPV-related lesions. (AmJ Pathol 1995, 146:589-598)