HEK293 cells were analysed for their ability to activate both IFNβ and ISRE reporters when stimulated with poly (I:C). These cells had shown a strong response to exogenous IFNα (Fig. 5.3). CMV-β-Gal was used as a control for transfection efficiency, since its activity was less sensitive than pRL-TK to external variables.
Figure 5.6 HEK 293 cells support both IFNβ and ISRE reporter assays. 2.5 x105 HEK293 cells were co-transfected in triplicate with 250ng pCI-neo, 25ng pcDNA3.1-HisB::lacZ and 225ng pLuc-IFNβ or 225ng pLuc-ISRE. 16 hours later cells were mock-transfected or transfected with 1µg poly (I:C). All cells
Reporter Treatment Sample Luciferase reporter activity (RLU) Adjusted β-Gal activity
(OD405nm) Ratio Average IFNβ- Luc 1 0.013 0.139 0.091 Mock 2 0.012 0.115 0.108 0.116 3 0.013 0.088 0.148 4 0.145 0.060 2.408 Poly (I:C) 5 0.116 0.072 1.604 2.140 6 0.153 0.066 2.300 ISRE- Luc 7 0.054 0.130 0.417 Mock 8 0.053 0.075 0.701 0.616 9 0.048 0.065 0.729 10 0.383 0.053 7.175 Poly (I:C) 11 0.437 0.038 11.387 8.686 12 0.348 0.046 7.494
Transfection of cells with poly (I:C) led to 18-fold and 15-fold increases in IFNβand ISRE reporter activities, respectively (Fig. 5.6). Most of the induction observed was due to a direct increase in luciferase reporter activity, asβ-galactosidase activity only varied by approximately 2-fold between mock-treated and poly (I:C)-treated cells (Table 5.2). This was in contrast to the 4-fold decrease in pRL-TK activity observed upon poly (I:C) treatment of U2OS cells (Table 5.1). This indicated that the β- galactosidase reporter was not significantly influenced by poly (I:C) treatment and could be deemed as a reliable indicator of transfection efficiency.
Table 5.2 HEK 293 cells support both IFNβ and ISRE reporter assays.
Values shown are from the experiment represented in figure 5.7. RLU: Relative light units. β-galactosidase activity is represented as the optical density at a wavelength of 405nm, and is adjusted to exclude the cellular background.
5.7 Discussion
The assays performed in this chapter were designed to identify a cell line that would respond effectively to the IFN inducer, poly (I:C). They were based upon transient transfection methodology, and therefore their success is highly dependent on the transfection efficiency of the cell line being used. If a cell line has a poor efficiency of transfection then general reporter activity across the cell population will be low. Consideration must also be taken that the stimulant used in these assays, poly (I:C) was also delivered to cells by transient transfection. Therefore, within a population of cells of poor transfection efficiency only a tiny subset of cells would be transfected with both the necessary reporter and the poly (I:C) required to induce it. In such a scenario only a small, if any, increase in reporter activity would be recorded. This could possibly explain why the IFNβ reporter responded relatively poorly to poly (I:C) in both HEp-2 and A549 cells (Fig. 5.2).
U2OS cells were initially found to support significant increases in both IFNβ and ISRE reporter activity, in contrast to that of either HEp-2 or A549 cells. However, upon closer inspection, much of the apparent induction seen was due to decreasing
Renilla luciferase activity after poly (I:C) treatment that was over 5-fold in some cases; this created an increase in corrected ISRE activity. Cell death could not account for this decrease, though could have reflected host cell translational shutoff induced by the IFN response. Renilla luciferase activity was also found to vary depending on the PML protein being expressed, suggesting that it was not independent of the experimental variables. Subsequent analysis of 293 cells, where a
β-galactosidase reporter was used as a transfection control, revealed that poly (I:C) treatment again decreased control reporter activity, though in this case the decrease was typically only <2-fold. Uncorrected IFNβ and ISRE reporter activity, however, gave strong responses to poly (I:C), suggesting that the relative induction was not
The mechanism underlying the differences between Renilla luciferase activity is unclear, though one possibility is that the autonomous HSV-TK promoter used to drive this reporter was being selectively down-regulated upon poly (I:C) treatment. The β-galactosidase reporter, in contrast, utilised a CMV immediate early promoter and therefore would not be subjected to the same regulatory mechanisms. Such a scenario could also explain why the Renilla luciferase activity was altered upon expression of various PML proteins.
As well as affecting translation, interferon has also been shown to exert anti-tumour properties (Gresser & Belardelli, 2002), and to enhance the p53 apoptotic response (Takaokaet al., 2003) . This has implications when performing IFN reporter assays in immortalised cell lines, as normal IFN responses may be intrinsically impaired. For example, Vero cells are known to lack the ability to produce IFN α due to a
mutation in the IFN β promoter (Emeny & Morgan, 1979). It is therefore not
inconceivable that the poor response to poly (I:C) observed in the majority of cell lines tested here may be due to deficiencies in the IFN signalling pathway upstream of IFNβ promoter activation. Indeed, U2OS cells have been suggested to possess deficiencies in IFN signalling (Mossman et al., 2000), which may be a contributing factor to their poor response to poly (I:C).
Aberrant IFN signalling pathways may also be responsible for the apparent lack of reporter induction by poly (I:C) in both A549 and HEp-2 cells. In particular, A549 cells were unable to induce ISRE reporter activity in the presence of exogenous IFN
α., though they did support induction of the IFNβ promoter by poly (I:C). Altogether, this suggested that A549 cells may possess deficiencies in IFN signalling
downstream of IFNβ production. It is of interest that a small induction of the ISRE
reporter was observed in A549 cells upon transfection with poly (I:C), indicating that
the cells may still respond to endogenously-produced IFNβ, but have lost the ability
to respond to exogenous IFNα. In contrast, the IFNβ reporter did not show any induction upon transfection of HEp-2 cells with poly (I:C). This would indicate a failure of IFN signalling upstream of IFNβinduction. However, further work would
also responded in a dose-dependent manner to the addition of exogenous IFN α, indicating that the IFN signalling pathway is functional within these cells. 293 cells are routinely reported to give very high transfection efficiencies, and in accordance with this, they also supported relatively strong expression of exogenous proteins, as observed in Fig. 3.8A. Altogether, this suggests that 293 cells are an ideal cell line for an analysis of the effect of different PML proteins on the IFN response that utilises reporter assays.