There is increasing evidence supporting DNA virus regulation of the cell adhesion and tumour suppressor protein, E-cadherin. We previously reported that loss of E-cadherin in human papillomavirus (HPV) type 16-infected epidermis is contributed to by the major viral proto-oncogene E6 and is associated with reduced Langerhans cells density, potentially regulating the immune response. The focus of this study is determining how the HPV16 E6 protein mediates E-cadherin repression. We found that the E-cadherin promoter is repressed in cells expressing E6, resulting in fewer E-cadherin transcripts. On exploring the mechanism for this, repression by increased histone deacetylase activity or by increased binding of trans-repressors to the E-cadherin promoter Epal element was discounted. In contrast, DNA methyltransferase (DNMT) activity was increased in E6 expressing cells. Upon inhibiting DNMT activity using 5-Aza-2′-deoxycytidine, E-cadherin transcription was restored in the presence of HPV16 E6. The E-cadherin promoter was not directly methylated, however a mutational analysis showed general promoter repression and reduced binding of the transactivators Sp1 and AML1 and the repressor Slug. Expression of E7 with E6 resulted in a further reduction in surface E-cadherin levels. This is the first report of HPV16 E6-mediated transcriptional repression of this adhesion molecule and tumour suppressor protein.

The importance of mitochondrial toxicity in drug-induced liver injury is well established. The bioenergetic phenotype of the HepaRG cell line was defined in order to assess their suitability as a model of mitochondrial hepatotoxicity. Bioenergetic phenotyping categorised the HepaRG cells as less metabolically active when measured beside the more energetic HepG2 cells. However, inhibition of mitochondrial ATP synthase induced an increase in glycolytic activity of both HepaRG and HepG2 cells suggesting an active Crabtree Effect in both cell lines. The suitability of HepaRG cells for the acute metabolic modification assay as a screen for mitotoxicity was confirmed using a panel of compounds, including both positive and negative mitotoxic compounds. Seahorse respirometry studies demonstrated that a statistically significant decrease in spare respiratory capacity is the first indication of mitochondrial dysfunction. Furthermore, based upon comparing changes in respiratory parameters to those of the positive controls, rotenone and carbonyl cyanide m-chlorophenyl hydrazone, compounds were categorised into two mechanistic groups; inhibitors or uncouplers of the electron transport chain. Overall, the findings from this study have demonstrated that HepaRG cells, despite having different resting bioenergetic phenotype to HepG2 cells are a suitable model to detect drug-induced mitochondrial toxicity with similar detection rates to HepG2 cells.

Human papillomavirus (HPV) infection is strongly associated with the development of anogenital neoplasia, particularly cervical cancer. It has been estimated that 99.7% of all cervical carcinomas are attributable to infection with HPV, and types 16 and 18 account for the vast majority of such cases. Both of these Ôhigh riskÕ HPV types encode the oncoproteins E6 and E7, which exert multiple effects on many proteins involved in cell-cycle regulation, including p53. The nuclear export protein inhibitor leptomycin B (LMB) has been shown to cause the nuclear sequestration of p53 in cervical carcinoma cells. We demonstrate that LMB induces apoptosis selectively at nanomolar concentrations in primary human keratinocytes (PHKs) expressing HPV oncogenes. Both monolayer and organotypic raft cultures of transduced PHKs were highly susceptible to treatment with LMB. By contrast, although LMB stimulated p53 accumulation in normal PHKs, no significant induction of apoptosis was detected on Western blots or immunostained monolayer/raft cells, or following pulsed exposure to the drug. Furthermore, topical application of lM concentrations of LMB to mouse skin was non-toxic. These data suggest that the topical application of LMB to HPV-infected intra-epithelial lesions may represent a specific and effective therapeutic strategy against HPV-associated anogenital neoplasia. ' 2007 Wiley-Liss, Inc.Key words: human papillomavirus; leptomycin B; keratinocytes; oncogenes; apoptosis Leptomycin B (LMB), a secondary metabolite produced by Streptomyces spp., 1 inhibits the export of proteins containing a nuclear export signal (NES) from the nucleus to the cytoplasm. This is achieved through inactivation of the nuclear export receptor protein CRM1 (exportin 1) by covalent modification. 2,3 LMB therefore promotes nuclear accumulation of NES proteins, including the tumour suppressor protein p53. 4,5 Furthermore, LMB has been shown to cause nuclear sequestration and reactivation of p53 in cervical carcinoma cell lines containing human papillomavirus (HPV) and this was associated with induction of apoptosis. 6,7 By contrast, LMB treatment resulted in the reversible cell-cycle arrest of both rat fibroblasts 8 and normal human primary fibroblasts. 5,9 Although the effect of LMB on CRM1 is well documented, the specific molecular mechanism(s) by which LMB induces cellcycle arrest and apoptosis still remain unclear.Since the discovery of LMB in the early 1980s, there has been considerable interest in the use of the compound as a cancer therapeutic. LMB (alternatively termed CI-940, elactocin or PD 114, 720) and its hydroxy analogue (PD 114, 721) were shown to possess potent activity against 7 different tumour models. 10 Based on these encouraging findings, a phase I clinical trial was initiated to assess the systemic administration of LMB. However, the trial was later terminated because of unwanted side effects. 11 Nevertheless, although there are difficulties in using LMB systemically, topical therapeutic use may be feasible.Cervical cancer is the ...

The aim of this study was to investigate the pathogenesis of combination ipilimumab and nivolumab-associated colitis (IN-COL) by measuring gutderived and peripheral blood mononuclear cell (GMNC; PBMC) profiles. We studied GMNC and PBMC from patients with IN-COL, IN-treated with no adverse-events (IN-NAE), ulcerative colitis (UC) and healthy volunteers using flow cytometry. In the gastrointestinal-derived cells we found high levels of activated CD8 + T cells and mucosal-associated invariant T (MAIT) cells in IN-COL, changes that were not evident in IN-NAE or UC. UC, but not IN-C, was associated with a high proportion of regulatory T cells (T reg). We sought to determine if local tissue responses could be measured in peripheral blood. Peripherally, checkpoint inhibition instigated a rise in activated memory CD4 + and CD8 + T cells, regardless of colitis. Low circulating MAIT cells at baseline was associated with IN-COL patients compared with IN-NAE in one of two cohorts. UC, but not IN-COL, was associated with high levels of circulating plasmablasts. In summary, the alterations in T cell subsets measured in IN-COL-affected tissue, characterized by high levels of activated CD8 + T cells and MAIT cells and a low proportion of T reg , reflected a pathology distinct from UC. These tissue changes differed from the periphery, where T cell activation was a widespread on-treatment effect, and circulating MAIT cell count was low but not reliably predictive of colitis.