Transcript profiling of putative NF-kB activators in wild-type and NFKB1-null HeLa cells
In future high-throughput transcriptomic studies, we propose that the use of cell lines nullizygous for important target genes of environmental chemicals will greatly facilitate the interpretation of gene expression profiles. To provide support for this concept, we generated transcript profiles of known or putative NF-kB activators in wild-type and NFKB1-null HeLa cells. Both cell lines were treated with interleukin 1 (IL1beta), tumor necrosis alpha (TNFalpha) or one of two chemicals identified in high throughput assays for NF-kB activity (chlorhexidine diacetate and carbocyanine). The profiles were generated using TempO-Seq targeted sequencing of ~3000 human genes. Almost all of the genes regulated by IL1beta and TNFalpha in wild-type cells were no longer altered in the NFKB1-null cells and approximately half of the genes regulated by Chlorhexidine diacetate and carbocyanine were dependent on NFKB1 for expression changes.  These studies demonstrate that many of the changes in gene expression after exposure to the putative NF-kB activators were NFKB1-dependent. Overall design: An initial range finding experiment was conducted in wild-type and the NFKB1-null HeLa cells, to determine the optimal concentration of the tested chemicals. Cells were plated in 12-well plates and incubated 20 hours. The media was replaced with dosing solutions containing DMSO (0.05%) or IL1beta (1 ng/mL), TNFalpha (5 ng/mL), chlorhexidine diacetate (50 µM), or carbocyanine (50 µM) in DMSO (0.05%).  After 6 hours of exposure, media was removed and cells were lysed in 0.3 mL Trizol, followed by RNA extraction. Gene expression changes in wild-type and NFKB1-null cells were evaluated using the human 1500+ Tempo-Seq platform (BioSpyder, Inc, Carlsbad, CA).