STUDY OF THE HUMAN PAPILLOMAVIRUS TYPE 16 (HPV-16) TISSUE SPECIFICITY

Abstract

Human Papillomavirus type-16 (HPV-16) infects specifically the mucosal epithelia, and is the papillomavirus type most frequently found in anogenital cancers, An 850 bp segment of the viral genome does not code for any genes and is termed the long control region (LCR). An enhancer of 400 bp in the LCR had been found to be cell-type specific (Gloss et al., 1987), as it functions as an enhancer in Hela (cervical carcinoma cell line) but not in MCF7 (breast carcinoma cell line), This was the first indication that the enhancer may contribute to the epitheliotropism of the virus. This thesis aims to confirm and extend the identification of the cell-type specificity of the HPV-16 enhancer in vitro and in vivo. The 400 bp enhancer was found to be strictly keratinocyte specific in tissue-culture experiments. It was active in all the epithelial cells tested, irrespective of their exact histological origin, It was also not species dependent, in that it was not selectively active in human keratinocytes. The range of epithelial cells tested included those derived from the cervix, mammary gland, colon, skin, lung, brain, bladder, liver (Hela, Siha, HT3, MCF7, Colo320HSR, Hacat, A549, H4, T24, HepG2, HD2). The enhancer was inactive in fibroblasts (MRHF, Bud8, C127, NIH3T3, Rat2) and other non-epithelial cell-lines, such as lymphoid cells (Daudi), and undifferentiated embryonal carcinoma cells (F9). It is observed that the 400 bp enhancer functions in both Hela and MCF7 cells, albeit to a lower extent in the latter. This therefore resolves the paradox to the claim that the viral enhancer was active in Hela and not in MCF7 cells, although both are keratinocytes. Smaller subfragments of the 400 bp enhancer (232 bp strong enhancer and 91 bp core enhancer) also showed similar keratinocyte-specific activity. Attempts to narrow down specific elements which contribute to this epithelial specificity of the virus, seem to point that the transcription factor NF1 might play a central role in this property. A human fibroblast cell-line (GM637), gave contradictarily high activity of the enhancer. Upon more detailed examination, it was discovered that these cells were probably contaminated with keratinocytes as they stained very strongly for keratins. This further implied the correlation between HPV expression and keratin expression. In the in vivo analysis, the 232 bp enhancer of HPV-16 was linked to simian virus 40 large-T oncogene driven by herpes simplex virus thymidine kinase promoter, and introduced as a transgene into the mouse genome, T-Ag thus acted as the tumorigenic marker, where its expression should be for the biggest part stimulated by the HPV enhancer. Aggressive tumors arose within 6 months of age of the mice, but were not specifically epithelial in origin, Brain tumors, osteosarcoma, pancreatic tumors and eye tumors predominated, The keratinocyte-specific property of the HPV-16 enhancer seen in tissue-culture experiments was not reflected in this test system, possibly either due to the background activity of the heterologous minimal promoter of the transgene, or the inherent 'oncogene specificity' of the T-antigen.