Molecular mechanisms of glucocorticoid-dependent oncogenesis and expression of human papillomavirus type 16 DNA

Mittal, Rakesh (1993) Molecular mechanisms of glucocorticoid-dependent oncogenesis and expression of human papillomavirus type 16 DNA. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Human papillomaviruses have been implicated as a causative agent in the etiology of many human cancers, especially cervical carcinomas. Our laboratory had previously shown that the presence of the steroid hormones, dexamethasone and progesterone, markedly enhances the transformation of primary rodent cells by HPV type 16 DNA in cooperation with the EJ-ras oncogene. This enhancement could have been direct, through a previously known glucocorticoid response element (GRE) located at nt position 7640 in the transcriptional regulatory region of the HPV 16 genome. Two additional GRE-like sequences were also found at nt positions 7385 and 7474. Alternatively, indirect mechanisms could be conceived through hormone-mediated expression of other cellular transcription factors which in turn modulate HPV gene expression. To address the role of the GRE located at nt position 7640, site-directed mutational analysis was performed. In transformation assays in cultured rodent cells and in transient CAT assays with the human cervical carcinoma cell line, HeLa, I found that loss-of-function mutations of this GRE retained the response to dexamethasone, indicating involvement of other factors. However, converting this GRE into the consensus sequence resulted in an increased frequency of transformation and a greatly increased expression in transient assays, indicating the role of this GRE also. Retention of a hormone response for the loss-of-function mutations led me to examine the role of the other two GRE-like sequences in transformation and gene expression. A series of single, double and triple mutants, containing different combinations of mutations in the three GREs, were tested in transformation and transient gene expression assays. The results showed that all three elements are individually functional and are required to observe any hormone effect in both assays. The two newly identified GREs were further characterized using synthetic oligonucleotides. Both GRE sequences were studied for their ability to respond to dexamethasone in transient CAT assays. In addition, specific DNA protein interactions were examined using several in vitro DNA-protein interaction assays. Results have shown that both GRE sequences respond to dexamethasone and bind specifically to a protein of 97 kDa, the molecular size of the native glucocorticoid receptor. -- As the GRE at nt position 7640 is a composite GRE with an overlapping AP-1 motif, that interacts with the cellular c-jun and c-fos transcription factors, the role of these cellular oncogenes in glucocorticoid-dependent expression of HPV 16 genes was also examined. The results demonstrated that c-jun conferred a positive response of dexamethasone-induced expression of viral genes, whereas, presence of c-fos inhibited this response. Interestingly, the composite GRE was responsive to dexamethasone only in the presence of c-jun, indicating the special significance of the composite GRE in HPV gene regulation. -- To examine if the GREs are functional in the principle host tissue for HPV 16 infection, primary human ectocervical cells were cultured and used to examine the effects of hormones on HPV 16 gene transcription. Viral RNA was examined using in situ hybridization, after transfecting either wild type or mutated HPV genomes into these cells. Viral transcription was observed for the wild type HPV genome only in the presence of these hormones and could be blocked by RU486, an anti-progestin, in a dose-dependent fashion. Constructs with all single or double GRE mutations also responded to both hormones, whereas, a triple mutated construct, with all three GREs disrupted did not support any detectable induction of viral transcription. Thus, steroid hormones appear to be essential for episomal expression of HPV genes in primary human cervical cells. This result also emphasizes the role of hormones in early stages of HPV infection where the majority of HPV DNA is found episomally.

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