Cytokeratin patterns of expression in human epithelial cell lines correlate with transcriptional activity of the human papillomavirus type 16 upstream regulatory region.

Abstract

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

Many different papillomaviruses have low transcriptional activity in spite of strong epithelial specific enhancers.

“…Independent of the differentiation state, the transcriptional environment of these cell lines repressed the transcription units of most PVs strongly, with moderate repression observed only in the case of HPV-11. Our observations are in line with those of other groups, as low transcriptional activity and correlations between transcription of HPVs and epithelial differentiation markers in cell culture and epithelial raft culture have been previously observed (Meyers et al, 1992 ;Cheng et al, 1995 ;Parker et al, 1997 ;Tergaonkar et al, 1997 ;Zhao et al, 1997). Low transcriptional activities of PVs observed in this study should not be regarded as an unexpected finding, as the persistence of PV genomes in small populations of slowly growing cells would be incompatible with high levels of viral oncogene expression.…”

Pleiotropic effects of human papillomavirus type 16 E6 oncogene expression in human epithelial cell lines.

“…Apart from their differences in genomic background, these cells differ in that HaCaT cells are merely immortalized and can be transformed by the ras oncogene (Skobe et al, 1997 ; A. Rangarajan and others, unpublished results), whereas C33A cells are derived from an invasive tumour. In addition, they represent two different stages of differentiation in that HaCaT cells express cytokeratin 14, a marker of basal keratinoctyes, and in contrast C33A cells resemble more differentiated Multiple cellular effects of HPV-16 E6 Multiple cellular effects of HPV-16 E6 keratinocytes in expressing cytokeratin 10 and not 14 (Tergaonkar et al, 1997). Thus, the differentiation status of the cell per se does not seem to modulate the effects of E6 reported in this study.…”

“…( f ) Cyclin A expression in HaCaT-Neo and HaCaT-E6 cells (i20 magnification). Immuno-stainings with primary antibodies (Santa-Cruz and gifts from N. Dyson, MGH Cancer Center) and detection was done as reported previously (Tergaonkar et al, 1997 ;Daniel et al, 1997). Over 85 % HaCaT-E6 cells showed cyclin A positivity in a low-power field.…”

Papillomavirus-Mediated Neoplastic Progression Is Associated with Reciprocal Changes in Jagged1 and Manic Fringe Expression Linked to Notch Activation

“…Transcriptional analyses were performed by transfecting the reporter constructs into the cell lines mentioned. The chloramphenicol acetyltransferase (CAT) reporter activity was normalized to the cytomegalovirus (CMV) promoter-driven Renilla luciferase activity as described earlier (50). In experiments involving inhibition of ligand-induced Notch signaling, 20 M GI (Calbiochem) was added 12 h posttransfection of the reporter constructs.…”

“…Immunohistochemistry was performed on organotypic raft sections to detect the levels of Notch1, Jagged1, HES1, and Manic Fringe proteins by using the antibodies mentioned above. The appropriate peroxidase-conjugated secondary antibodies (Molecular Probes) were used, and DAB precipitation was performed as previously reported (50).…”