Analysis of the role of glucocorticoid response elements (GREs) of human papillomavirus type 16 through consensus mutations of GREs

Khare, Suvarnalatha (1995) Analysis of the role of glucocorticoid response elements (GREs) of human papillomavirus type 16 through consensus mutations of GREs. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (21MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

The association of certain papillomaviruses with cancer of the uterine cervix has been established unequivocally by previous studies. In addition, steroid hormones, especially glucocorticoids and progesterone, have been implicated as an important cofactor in the oncogenesis. In this study, the role of glucocorticoid hormones in human papillomavirus type 16 (HPV16) expression and in the immortalization of cervical cells by HPV16 was analyzed. -- To demonstrate the direct effect of the hormones on expression, the three glucocorticoid response elements (GREs) in the HPV16 regulatory region were mutated to the consensus GRE (cGRE) sequence, GGTACA(N)₃TGTTCT. The effect of these mutations on expression was assayed using the chloramphenicol acetyl transferase (CAT) reporter gene, which was placed under the control of the HPV16 enhancer with the cGREs, in pBLCAT2 vector. Activity of the CAT from these constructs was assayed in the HeLa cervical carcinoma cells. The CAT assays revealed that both single cGRE and triple cGRE mutations enhanced the expression of the reporter gene in the presence of glucocorticoid dexamethasone (dex). Similar results were obtained in the CAT assays when these constructs were transfected into primary baby rat kidney (BRK) epithelial cells. -- To examine the molecular involvement of the GREs in the enhanced in vivo CAT activities from cGRE constructs, assays for DNA-protein interaction were performed in vitro using 22 base pair oligomers, representing the wild type GREs or cGREs at nucleotide (nt) 7385 and nt 7474, and HeLa whole cell protein extracts. Binding with the 22-mer representing the nt 7474 cGRE sequence in the mobility shift assays was more, in comparison with the wild type GRE 22-mer. A moderate difference between the binding ability of nt 7385 GRE and the cGRE 22-mers was evident. The results of UV crosslinking indicated that a HeLa cell protein of approximately 96 kDa, the size of the native glucocorticoid receptor (GR), interacted with the different GRE 22-mers. Southwestern blot assays using these 22-mers also suggested the interaction of the cGRE sequences with a protein, possibly the GR, which is known to be present in HeLa extracts. -- The HeLa cervical cell-pBLCAT2 system was an efficient and quantitative model for dex-induced expression, possibly mediated by GR. To advance to a homologous promoter and the natural target of HPV infection, whole HPV16 constructs and primary cultures of ectocervical cells were chosen. The effect of the cGRE mutations on the expression of E6 and E7 viral oncogenes in these cells was analyzed by the sensitive in situ hybridization assays. Strong hybridization signal for the E6-E7 oncogenes was displayed in the presence of dex, when the RNA from the cells transfected with the HPV16 genome construct containing cGREs, was analyzed. Next, these ectocervical cells, which are the appropriate cell type and the natural target of steroid hormone action, were chosen for stable transfection of the whole HPV16 genome construct with triple cGREs. An immortalized population of cells, designated HEC-16cGRE, was established. These cells were morphologically distinct from the parental ectocervical cells. Analysis of the high molecular weight DNA in HEC-16cGRE cells by Southern blot indicated the presence of HPV16 DNA in an integrated form. The integrated HPV16 DNA was transcriptionally active, as shown by Northern blot assays which showed 4.5 and 2.3 kb transcripts, that contain mostly E6-E7. Interestingly, the viral RNA levels were about 2-fold higher in HEC-16cGRE than in HEC-16, which contains HPV16 with wild type GREs. These cells were not tumorigenic, as shown by soft agar assays and injection into nude mice. Further, the HPV16 genome construct with triple cGREs exhibited greater transformation of BRK cells with activated ras oncogene than the wild type HPV16 genome. -- In conclusion, these results demonstrated an increase in expression and transformation from triple cGRE constructs. The enhanced expression was correlated with the specific protein-binding to the GREs. The HEC-16cGRE cells generated from HPV16 with cGRE mutations could be a valuable tool to test the models proposed earlier for HPV and hormone-mediated oncogenesis.

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