Agonist responses in HeLa cells transduced with the EGFP-GCCR adenoviral vector
Figure 5 shows agonist responses for a range of glucocorticoid compounds and other steroid analogues. The data show differential potency of agonists, with the expected high potency of dexamethasone (mean nucleus to cytoplasm ratio 6.33, SD 1.154) and prednisolone (mean 6.79, SD 0.938) at the GCCR. The high response to RU486 (mean 4.05, SD 0.348), which is reportedly an antagonist, appears to support the observation that RU486, in this system, is functioning as an activator of translocation, as has been noted before (2). Use of the EGFP-GCCR fusion protein enables facile identification of this phenomenon.
Function of gene sensors: Adenoviral transduction vs stable cell lines
The use of adenoviral vectors to generate the transient expression of key genes can avoid the laborious and time-consuming establishment of stable cell lines. It is therefore important to demonstrate that the functionality of the transiently expressed gene is comparable, within certain criteria, to equivalent genes expressed in stable cell lines.
Figure 6 summarizes the response to dexamethasone of target HeLa cells transiently transfected, at a range of MOI, with the EGFP-GCCR adenoviral vector. This was compared to equivalent responses in stable HEK 293 cell lines expressing GFP-GCCR. The data show MOI-dependent functional responses in target HeLa cells that have been transiently transfected with the EGFP-GCCR adenoviral vector. Maximum response, as measured by the nuclear to cytoplasm intensity ratio, was at 40 MOI. However, at all MOI values tested in this experiment, EC50 values calculated for the corresponding assays were comparable, indicating similar sensitivities to the detection of agonists across the range of MOI values employed.