References

Notes: Expression of the early genes of human papillomavirus type 16 (HPV16) is known to be highly restricted to epithelial cells. This report describes the molecular basis of HPV16 epitheliotropism at the transcriptional level. A series of 5'-end deletion mutants of the long control region (LCR) located upstream of the HPV16 early genes were tested in a transient expression assay using various human epithelial and fibroblastic cell lines. The specific region termed the cell-type-dependent regulatory element (CTRE) was found to function as an enhancer in some of HPV-associated human epithelial cell lines, but not at all in HPV-free human epithelial cell lines. No LCR-associated enhancer activity was detectable in fibroblastic cell lines. The CTRE was mapped at a distinct region upstream from the previously proposed proximal keratinocyte dependent enhancer (KD). The CTRE augmented transcription initiated from the autologous P97 promoter as well as from a heterologous promoter in a orientation- independent manner. Within the CTRE, there are three copies of the consensus nuclear factor-1 (NF1) binding site. Using CTRE containing mutations in each of the NF1-sites, it was demonstrated that all of these NF1-sites were necessary for a full enhancer activity in the HPV-associated human epithelial cell line. This suggests that the CTRE plays a critical role in the cell-type specific expression of HPV16-early gene at the level of transcriptional regulation
Reference ID: 3938

Notes: Laboratory of Molecular and Cell Biology, Kanagawa Cancer Center Research Institute, Yokohama, Japan 0022-1317 ENGLISH ENGLAND Cytogenetic abnormalities associated with human papillomavirus (HPV) type 16 were studied using primary human and mouse epidermal keratinocytes. The E7 transforming gene of HPV-16 was found to induce chromosome duplication in epidermal keratinocytes; little or no detectable chromosome disorganization was associated with the function of the E6 gene. These results suggest that the E7 gene-linked cytogenetic effect reflects HPV-16-associated pathogenicity in the early phase of transformation E6; E7 JOURNAL-ARTICLE 0; 0; 0; 0; 0; 0 91311409 9110
Reference ID: 4915

Notes: Laboratory of Molecular and Cell Biology, Kanagawa Cancer Center Research Institute, Yokohama, Japan 0022-538X ENGLISH UNITED-STATES We have found that epidermal growth factor (EGF) elicits negative regulation of human papillomavirus type 16 (HPV-16) E6/E7 at the mRNA level in the HPV-16-immortalized human keratinocyte cell line (PHK160b). This down-regulation of HPV-16 E6/E7 expression was achieved when the cells were stimulated to proliferate with the concomitantly enhanced c-myc expression by EGF in a dose-dependent manner. By using partly synchronized PHK160b cells, negative and positive regulations of the HPV-16 E6/E7 expression was correlated to EGF-linked cell cycle events in this particular human keratinocyte cell line. In order to study transcriptional control mechanisms of the HPV-16 E6/E7, transient expression assays were performed with CAT expression plasmids that the transcription could be directed by the 5'- deleted HPV-16 long control region (LCR) including the virus P97 promoter. We demonstrated that the HPV-16 LCR contained EGF- responsive elements and that a predominant silencer activity was mapped in the proximal 124-bp region (EGFRE) of the LCR. This restricted LCR region had significant influence on HPV-16- homologous promoters in lowering the CAT expression in the presence and absence of EGF. EGFRE was thus considered to be a conditional transcription-controlling element on HPV-16 E6/E7 expression in this EGF-responsive human keratinocyte cell line. This suggests that specific sequences in the LCR play a critical part in the EGF-induced down-regulation of E6/E7 expression at the transcriptional level. Since the results obtained from the transient expression assay agreed with the mode of expression of the endogenous HPV-16 E6/E7, the present study strongly suggests that the transcriptional regulation of the HPV-16 E6/E7 oncogene is mediated by growth-related specific cellular factors interacting with HPV-16 LCR elements JOURNAL-ARTICLE EC 2.3.1.28; 0; 0; 62229-50-9 91162752 9106
Reference ID: 4916

Abstract: We have re-evaluated transforming activities of HPV 16-E6 and -E7 in terms of growth modulation, extended life span and immortalization of normal human keratinocytes in vitro. Effecient transfection of E6 and E7 genes by electroporation stimulated cell proliferation of primary human keratinocytes and potentiated to form micro- and macro-colonies shortly after the transfection. The E7 gene alone induced an apparent extended life span by exceeding 50 population doublings (PDL) but rarely greater than 150PDL expressed high levels of the E7 protein, suggesting that E7-associated cellular events are necessary for the extended life span but almost all E6-macrocolonies could not be expanded to grow after processing in mass culture. Thus, the transforming activity of E6 appeared to be much weaker than that of E7. Co-transfection of E6 and E7 genes produced an immortal cell line, but the efficiency was low. The results suggest taht mechanistic role of E6 and E7 genes are different and complimentary in part in the immortalization of normal human keratinocytes in vitro. Cellular phenotypes of all immortalized cell lines changed progressively in vitro mimicking in vivo tumor development, indicationg that, in addition to the E6 and E7 functions, more critical cellular events are required for the immortalization and malignant transformation of human keratinocytes.
Reference ID: 4923