At the same time this cell line has a number of additional favourable properties (e.g. origin, adhesion, growth characteristics) and which strongly contribute to its suitability. Most important however, is the fact that the type of reporter constructs that we have used in the past (and also in this cell line) is generally characterized by very low backgrounds and thus consequently can yield high fold-induction levels provided the signalling pathways necessary for stimulation of the corresponding reporter are operational in the cell used. As the NF-KappaB signalling pathway is quite active in 293 cells also this condition is fullfilled in this case. Finally it is absolutely necessary that the stable cell lines engineered are indeed stable and contain the original constructs after substantial subculturing. Also is this aspect 293 cells are generally not disappointing.
On the basis of the results obtained so far with this engineered stable 293 cell line we are confident that we have developed a robust and reliable system which can be used for high-throughput screening for novel anti-inflammatory glucocorticoids.
First stable NF-kappaB luciferase reporter 293 (human embryonic kidney) cell lines were engineered by transfection of the luciferase reporter 4XNF-kappaB-E1B-tata-Luc carrying four kappaB sites from the HIV-LTR in combination with Pgk-HYG. After selection by hygromycin for twelve days and subsequent clonal isolation a number of stable cell lines were tested for reporter activity by the addition of TNF-alpha to the cultures and measuring luciferase activity. From the positive responders a limited number was selected for the next round of stable transfection with human glucocorticoid receptor (hGR) on the basis of the maximum response to TNF-alpha. To obtain double transfectants pSG5-hGR was transfected together with pGEM-NEO. Stable transfectants were selected and cloned after culturing for twelve days in the presence of both hygromycin and G418. Two stable clonal cell lines (#7 and #14) were selected on the basis of pre-screening for represssion by dexamethasone of reporter activity induced by TNF-alpha. The presence of GR was established by performing Western blotting using an antibody against hGR. The isolation of these stable double transfectants provided us the possibility to study molecular crosstalk betweeen GR and NF-kappaB in stable cell lines in which both pathways are operational and a an easy read-out of the system is provided by luciferase activity which can be measured in 96-well plates using the Luclite luciferase reporter assay kit (Packarad Instruments, Meridan,CT, USA) in a Topcount liquid scintillation counter (Packard Instruments, Meridan, CT, USA).
In both selected cell lines high levels of repression (80-90%) of TNF-alpha stimulated NF-kappaB reporter activity could be obtained routinely (after 24h of culture) by the synthetic glucocorticoid dexamethasone (0.01 mM), while the glucocorticoid antagonist RU486 gave around 50 % repression at 0.1 mM. The pure antagonist ZK988299 however gave no repression. In control cell lines without hGR and with the reporter only no repression could be observed. We also tested the ability of two other synthetic glucocorticoids to repress NF-kappaB reporter activity. Also beclomethasone dipropionate and budenoside are about as potent repressors of NF-kappaB reporter activity in this system as dexamethasone. These results clearly indicate that these reporter cell lines can be used in (high-throughput) screening assays for compounds exhibiting inhibition of the NF-kappaB pathway via GR and thus are very useful for the development of novel anti-inflammatory glucocortcoids. We are not aware of the existence of similar reporter cells anywhere.
