A comparison of the CAT reporter activity of a plasmid which contains the 232 bp epithelial specific enhancer alone with that of plasmids which contain additional sequences from the human papillomavirus type 16 ( HPV-16) upstream regulatory region (URR) revealed two markedly different patterns, in an analysis of six human epithelial cell lines. In HeLa, C33A and SiHa, the CAT reporter activities of all the constructs were comparable. In contrast, in CaSki, HK2bE6-E7 and HaCaT we detected very low levels of CAT reporter activity using the constructs with the additional HPV-16 URR sequences. The ability of HPV-16 E2 to transactivate a construct with 2 E2 binding sites also differed markedly and showed the same pattern. Cytokeratin staining revealed a correlation between cytokeratin 10 and 14 expression and the transcriptional differences observed. We also found alterations in the activity of one of the constructs on altering the growth conditions of the HaCaT cell line.Human papillomavirus type 16 (HPV-16) genomes are present in a very large proportion of cervical cancers and the E6 and E7 oncogenes of the virus are believed to be the major transforming genes (for review see zur Hausen, 1996). The non-coding region of HPV-16 is usually referred to as the upstream regulatory region (URR) since it modulates the activity of the p97 promoter which controls transcription of the E6 and E7 oncogenes. In situ hybridization studies on clinical infections (Durst et al., 1992 ;Stoler et al., 1989) and virion generation efforts using raft culture systems have emphasized (Dollard et al., 1992) both the epithelial specificity and the tight link between the regulation of papillomavirus transcription and the differentiation of epithelial cells. In addition, during the progression to higher grade lesions and

Two human papillomavirus (HPV)-negative epithelial cell lines, HaCaT and C33A, were transfected with HPV-16 E6 and analysed for functional consequences which are relevant to invasive tumour progression. After transfection with E6, both cell lines invaded collagen matrices, in contrast to vector-transfected control cells. The E6-expressing cells showed a marked increase in expression of the β1 integrin subunit, with no or relatively minor alterations in the levels of a range of integrin subunits. In addition, the epithelial cell lines expressing E6 displayed resistance to apoptosis generated by serum starvation. This resistance is comparable to that generated by ras and is not generated by HPV-11 E6 or HPV-16 E7. Both C33A and HaCaT cells have mutations in the p53 loci and hence these functional consequences of E6 are probably independent of wild-type p53 function.The persisting presence of human papillomavirus (HPV) types 16 and 18 E6 and E7 oncogene expression is a hallmark of most cervical tumours (zur Hausen, 1996). To elucidate the function of these two oncogenes, several groups have attempted to identify host proteins that interact with the products encoded by these two oncogenes. The bestdocumented function of HPV-16 E6 is the ability to bind (Storey et al., 1998 ;Werness et al., 1990) and degrade the p53 protein (Scheffner et al., 1990). More recently, largely using the yeast two-hybrid system, several host proteins, which include E6BP, paxillin, hDLG and AP-1, have been identified as interacting with the E6 product of different papillomavirus types (Chen et al., 1995 ;Kiyono et al., 1997 ;Lee et al., 1997 ;Tong et al., 1998 ;Tong & Howley, 1997 ;Vande Pol et al., 1998). In this report, we have explored the phenotypic consequences of E6 expression per se in epithelial cell lines. In general, transformed cells are generated following the cumulative effects of changes in apoptosis induction, cell cycle control, motility, integrin expression and function. These processes are linked in intricate feedback loops and the deregulation of the feedback controls during tumorigenesis is currently under investigation by several groups (Meredith & Schwartz, 1997 ;Schwartz, 1997). In this report, we have focused on an analysis of the role of the HPV-16 E6 oncogene in altering motility, integrin patterns and generating resistance to apoptosis induction in human epithelial cell lines.We have used two HPV-negative cell lines, C33A (Crook et al., 1991), a cervical tumour-derived cell line, and HaCaT (Lehman et al., 1993), a spontaneously immortalized epithelial line. Using plasmid LXSN-16E6 (Etscheid et al., 1994), we generated HaCaT-E6 and C33A-E6, which are HaCaT and C33A cells that constitutively express HPV-16 E6. Our approach using pooled transfectants is similar to that used in a recent study undertaken to analyse the role of cdc42 and Rac1 in integrin-mediated cell motility and invasiveness through PI(3)K (Keely et al., 1997). LXSN-neomycin-expressing cells, i.e. HaCaT-Neo and C33A-Neo, were used as controls in all e...