Tissue culture methods can strongly induce immediate early gene expression in retinal pigment epithelial cells.
Expression and activation of AP-1 transcription factor proteins is stimulated by diverse physiological factors including cytokines, growth factors, and various cell stressors. The studies presented here arose out of observations in our laboratory that there was rapid and significant induction of immediate early gene (IEG) transcription in "control" retinal pigment epithelial cell (RPE) cultures following media changes. To clarify whether routine in vitro manipulations have the potential to induce the expression of transcription factors and non-transcription factor genes, we performed quantitative PCR studies on RPE cells in culture following various cell rinse conditions. Our studies showed that there is rapid and dramatic induction of FosB, JunB, and EGR-1 transcription within 1 h following media aspiration and rinsing of confluent cells in vitro. The induction of these genes ranged from 32- to 256-fold following a buffered saline or media rinse. Modifying the rinse conditions and media used can eliminate this early response; however, a significant effect is still seen for FosB and JunB, 4 h after rinsing. The response was not seen with non-transcription factor genes and can be eliminated for most of the genes using a non-rinsing method. These studies demonstrate that rinsing cells in culture has the potential to profoundly affect subsequent analyses of gene expression and must be carefully controlled.
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Huaitao
H
Yang
Xiuying
X
Yang
John C
JC
Lang
Edward
E
Chaum
D002460:Cell Line; D051766:Early Growth Response Protein 1 / Q000378:metabolism; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013997:Time Factors; D046509:Tissue Culture Techniques / Q000379:methods*
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C064269:FOSB protein, human; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1