Abstract
Human papillomaviruses are DNA tumor viruses. A persistent infection with high-risk HPV types is the necessary risk factor for the development of anogenital carcinoma. The E6 protein is a viral oncoprotein that directly interacts with different cellular regulatory proteins mainly affecting the cell cycle, cellular differentiation and polarization of epithelial cells. In dependency of the phylogenetic classification of HPV different interaction partners of E6 have been described. The Notch pathway seems to be one common target of HPV, which can be up or down regulated by different E6 proteins. Our novel triple fluorescence flow-cytometry-based assay allows a semi-quantitative comparison of the E6 proteins´ effect on the Notch pathway using a Notch-responsive reporter plasmid. As a result, all E6 proteins of beta-HPV repressed the Notch reporter expression, of which HPV38 E6 showed the greatest repression potential. In contrast, alpha-HPV E6 of HPV16, activates the reporter expression most significantly, whereas E6 of HPV31 and low-risk HPV6b showed significant activation only in a p53-null cell line. Interestingly, HPV18 E6, with the second highest carcinogenic risk, shows no effect. This high divergence within different genus of HPV is important for targeting the Notch pathway regarding a potential HPV therapy.
Highlights
We developed a flow-cytometry-based assay to analyze P-HES1 activity using three different fluorescent proteins reporting (I) the transfection of the activator plasmid, human Notch 1 intracellular domain by EGFP co-expression, (II) the intracellular amount of the modulator by N-terminal fusion of mTagBFP2 and (III) the promotor activity by DsRed[2] expression (Fig. 2)
C33A and H1299 cells showed less than 0.2 ± 0.06% and 0.02 ± 0.003% DsRed[2] expressing cell population, respectively (Fig. 4), indicating that the constitutive and endogenous Notch signaling is low and thereby not triggering P-HES1 regulated reporter expression sufficiently
C33A and H1299 cells co-transfected with the activator plasmid showed a significant increase in the DsRed[2] expressing cell population, which was at least tenfold above the background signal of DsRed[2] expressing cell population without exogenous Notch intracellular domain (NICD). Both cell lines clearly show an activation of the Notch pathway by exogenous NICD with very low background of endogenous P-HES1-activity (Fig. 4b and c)
Summary
Co-transfection efficiency of plasmid DNA or low expression yields and high turnover rates of the modulatory proteins can deteriorate the signal-to-noise ratio of the reporter assay because of e.g. the high number of cells negative for the modulatory protein. To reduce this background, flow-cytometry is feasible to identify cells, which express the modulatory protein genetically fused to FPs, and monitor the reporter activity only in these positive cells. The effect of modulatory factors such as drugs, viral or cellular proteins on the Notch signaling pathway can be monitored by measuring the HES1 promotor activity (P-HES1).
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