2, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany
Inducible gene expression is a powerful tool for basic research, gene therapy and biotechnology, whose utility depends in part on consistent levels of induction regardless of metabolic status or physiological context. Here we examined the inducibility of the ecdysone receptor–based RheoSwitch mammalian inducible expression system in proliferating cells and in cell cycle–arrested cells. We found that both contact inhibition and growth arrest subsequent to serum deprivation dramatically reduced the levels of induction of reporter genes that could be achieved in 3T3 fibroblasts but in not NMuMG mammary epithelial cells. These data have implications for the use of the RheoSwitch system in inducible gene expression applications.
The ability to induce specific ectopic expression of a transgene, while not directly affecting expression of endogenous genes, has a wide range of important applications. These include basic research aimed at identifying the function of genes in cells or animals (1), treatment of diseases through targeted gene therapy (2), and the biotechnological production of useful biomolecules (3). Inducible gene expression systems need to fulfill a variety of criteria, including very low basal expression; highly specific direct induction of only the inducible gene; dose-dependent response to the inducing reagent; and consistent levels of induction regardless of metabolic state or physiological context. Several inducible promoter systems have been developed, including tetracycline-regulatable promoters, promoters activated by rapamycin-induced heterodimerization of FKB12 and FRB derivatives, and hormone-sensitive promoters such as those based on the progesterone antagonist mifepristone and the anti-estrogen Tamoxifen (reviewed in Reference (4).
One of the most promising inducible gene expression systems uses the insect steroid hormone ecdysone. The ecdysone receptor (EcR) forms a heterodimer with ultraspiracle (USP), the insect ortholog of the vertebrate retinoid X receptor (RXR). In the absence of ligand, the EcR/USP complex binds to its recognition element in the promoter of EcR-responsive genes and suppresses their transcription. Upon binding of ecdysone, conformational changes in the EcR convert the complex into a transcriptional activator (5). Insect EcR can functionally heterodimerize with vertebrate RXR. Potent inducible gene expression systems for mammalian cells have been developed using these components. A two-hybrid format (RheoSwitch), based on a Gal4-DNA binding domain fused to the EcR and a VP16-activation domain fused to the RXR, exhibits very low basal promoter activity and up to 10,000-fold induction in various mammalian cell types (6,7). Furthermore, it is responsive to synthetic non-steroidal ecdysone agonists such as the diacylhydrazine RSL1 (7,8).
Density-dependent inhibition of cell growth is a property of many types of non-transformed cells cultured in vitro, while loss of contact inhibition is a hallmark of malignant tumor cells (reviewed in Reference 9,. Contact inhibition is caused by cell cycle arrest (10). The molecular basis for contact-mediated growth control is poorly understood, but could be highly relevant for the therapeutic targeting of cancer cells. In addition to cell contact, quiescence can also be induced by serum deprivation. With these observations in mind, we set out to determine the utility of the RheoSwitch system for inducing expression of genes in contact-inhibited cells and in cells in serum deprivation–induced quiescence.Materials and methods Cell culture
The fibroblast RheoSwitch Cell Line NIH3T3–47 (New England Biolabs, Frankfurt, Germany) was cultivated in DMEM supplemented with 10% donor calf serum (DCS). NMuMG breast epithelial cells (11) were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and 10 μg/mL insulin. For contact inhibition experiments, the cells were seeded sparsely (2.5×103 cells/cm2) or densely (1×105 cells/cm2) and cultivated for 56 h. For serum deprivation experiments, cells were cultivated in medium containing 0.3% serum for 48 h.BrdU immunofluorescence
NIH 3T3 and NMuMG cell were pulsed with 10 μM BrdU (Roche Diagnostics, Mannheim, Germany) for 3 h and 2 h, respectively. Cells were subsequently fixed with 4% paraformaldehyde then permeabilized with 1% NP40/PBS. After treatment with DNase (Fermentas, St. Leon-Rot, Germany) and blocking with 10% DCS in PBS, BrdU incorporation was detected using a monoclonal rat anti-BrdU antibody [BU1/75 (ICR1), Abcam, Cambridge, UK] and an anti-rat Alexa Fluor 546 secondary antibody (Invitrogen/Molecular Probes, Inc., Eugene, OR, USA). Nuclei were stained with 10 μg/mL DAPI (Sigma-Aldrich, Taufkirchen, Germany). Samples were mounted with Mowiol 4–88 (Roth, Karlsruhe, Germany).Plasmid constructs
The pNEBR-R1 regulator plasmid encoding the RheoReceptor-1 and RheoActivator protein, and the pNEBR-X1-Hygro plasmid of the RheoSwitch inducible system were purchased from New England Biolabs (Ipswich, MA, USA). The firefly (Photinus pyralis) luciferase gene from the pGL3-Basic-Vector (Promega, Mannheim, Germany) was inserted into the pNEBR-X1 plasmid to create the firefly-pNEBR-X1-Hygro construct. The full-length Pip92 cDNA was also cloned into the pNEBR-X1-Hygro vector to create the Pip92pNEBR-X1-Hygro plasmid. The Ubi-Renilla construct (pRL-CMV) was provided by Olivier Kassel (Forschungszentrum, Karlsruhe, Germany) (12).