Paulmurugan and Gambhir 1010.1073/pnas.0607385103 |
Fig. 6. (a) Schematic diagram of vectors constructed to determine the orientation of N- and C-split RLUC fragments for efficient ER ligand-induced complementation. (b) Orientation-specific split RLUC complementation in response to the ER antagonist tamoxifen. 293T cells transiently transfected to express the intramolecular folding sensor N-RLUC-hER281-595-C-RLUC showed significantly higher levels of tamoxifen-induced RLUC complementation than the cells expressing C-RLUC-hER281-595-N-RLUC (P < 0.001).
Fig. 7. Chemical structures of different ER ligand agonists, antagonists, partial agonist, partial antagonist, and non-ER ligands used in the study.
Fig. 8. Comparison of ligand-induced split RLUC complementation under different assay conditions. The 293T cells were transiently transfected with the vector expressing the intramolecular folding sensor N-RLUC-ER281-549-C-RLUC and treated with ER ligands 17b-estradiol (E2), diethylstilbestrol (DES), and 4-hydroxytamoxifen (4-OHT) or carrier control (DMSO). RLUC activity was determined 18 h after treatment by luminometer assays with cell lysates (a) or bioluminescence imaging by optical CCD camera with cell lysates (b) and intact cells (c) after the addition 1 mg of the RLUC substrate coelenterazine per well.
Fig. 9. Comparison of ligand-induced RLUC complementation in 293T cells expressing the intramolecular folding sensor with wild-type and mutant hER (G512T). 293T cells transiently transfected with the indicated constructs were treated with ER ligands (1 mM) for 18 h before RLUC assay as described above. The G521T mutation in hER led to selective reduction of split RLUC complementation in response to the ER ligand 17b-estradiol. The experiment was repeated three times, and RLUC activity was expressed as mean ±SE.
Fig. 10. Comparison of mouse ER and human ER with analogous glycine mutations in ligand-induced split RLUC complementation. 293T cells expressing the intramolecular folding sensor (N-RLUC-ER-C-RLUC) containing the mutant human ER281-595/G521T (hER) was compared with that of the mutant mouse ER281-599/G525R (mER) for split RLUC complementation induced by different ligands (1 mM) as indicated. RLUC activity was determined at 18 h after treatment as described in Supporting Materials and Methods. The experiment was repeated three times, and RLUC activity was expressed as mean ±SE.
Fig. 11. ER intramolecular folding sensors with split FLUC enzyme fragments constructed for imaging ligand-induced intramolecular folding and ligand-induced intramolecular folding that distinguishes ER ligands in living animals.
Fig. 12. Intramolecular folding sensor with FLUC fragments showing ligand-induced complementation with different ER ligands.
Fig. 13. Bioluminescence imaging to differentiate ER ligands in living animals. Shown is optical CCD camera imaging of 293T cells stably expressing the intramolecular folding sensor (N-FLUC-mutant-hER281-549/G521T-C-FLUC) in living female nude mice before and after treatment with agonist diethylstilbestrol (DES), antagonist 4-hydroxy tamoxifen (4-OHT) (20 mg per mouse), or adjuvant. The corresponding graph shows the quantitative result from the images. The error bars represent SEM of four determinations.
Supporting Materials and Methods
Chemicals, Enzymes, and Reagents.
Restriction and modification enzymes and ligase were purchased from New England Biolabs (Beverly, MA). TripleMaster TaqDNA polymerase (Brinkmann Eppendorf, Hamburg, Germany) was used for the PCR amplification of different fragments of the reporter gene rluc and human estrogen receptor a (ERa) (hERa/HE0) (1). The plasmid pCMV-hRL (Promega, Madison, WI) was used as template for the amplification of rluc fragments. Different ER antagonists and agonists include tamoxifen, 4-hydroxytamoxifen, raloxifene, diethylstilbestrol, 17b-estradiol, genistein, anticancer drugs cisplatinum and epigallocatechin gallate (from green tea), DMSO, and antibiotics for bacterial cultures were purchased from Sigma (St. Louis, MO). Lipofectamine transfection reagent was purchased from Invitrogen (Carlsbad, CA). The plasmid and DNA gel extraction kits were purchased from Qiagen (Valencia, CA). Coelenterazine was from Nanolight (Pinetop, AZ). Bacterial culture media were purchased from BD Diagnostic Systems (Sparks, MD). All cell culture media, FBS, and the antibiotics streptomycin and penicillin were purchased from Invitrogen. The antiestrogen drug ICI182,780 was purchased from Tocris Cookson (Ballwin, MO). The custom oligonucleotides synthesized by the Stanford Protein and Nucleic Acid (PAN) facility were used as primers for PCR amplification. The site-directed mutagenesis kit from Stratagene (La Jolla, CA) was used for constructing mutants of the ligand-binding domain of human ER. The sequences of all vector constructs were verified by sequencing at the Stanford PAN facility. The ERa antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Construction of Different Plasmid Vectors.
The plasmid vector pcDNA-N-rluc-FRB from a previous study (2) was used as the starting vector. The C-rluc fragment was PCR-amplified by using forward primer designed with the BamHI restriction enzyme site and the reverse primer with stop codon and the XhoI restriction enzyme site. The amplified fragment was digested with respective enzymes and inserted into the starting vector digested with BamHI and XhoI, which releases out FRB fragment (pcDNA-N-rluc-) as pcDNA-N-rluc-C-rluc. The PCR-amplified fragments of human ER of different lengths were amplified by using the forward and reverse primers designed with the BamHI restriction enzyme site on either side were inserted into the same enzyme-digested and dephosphorylated pcDNA-N-rluc-C-rluc backbone as pcDNA-N-rluc-ER-C-rluc. By replacing the N- and C-RLUC fragments with the newly identified firefly luciferase (FLUC) fragments we constructed different sensors mentioned in Fig. 10. The Stratagene site-directed mutagenesis kit was used to construct the intramolecular folding sensor with the mutant forms of human ER.Cell Cultures
. All cell lines used for this study were purchased from American Tissue Type Collection (Manassas, VA). Human 293T embryonic kidney cancer cells were grown in MEM supplemented with 10% FBS and 1% penicillin/streptomycin solution. The MCF-7 human breast cancer cells were grown in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. ER-negative MDA-MB-231 cells were maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.Cell Transfection and Luciferase Assay.
Transfections were performed in 80% confluent 24-h-old cultures of 293T, MCF-7, and MDA-MB-231 cells by using Lipofectamine. For transient transfection studies, 200 ng of DNA per well was used in 12-well culture plates following the manufacturer's instructions. A total of 10 ng of FLUC plasmid DNA per well was cotransfected to normalize for transfection efficiency. The cells were assayed after 18 h of incubation at 37°C with 5% CO2. The luminometry assay for RLUC activity was performed as previously described (3). In brief, the cells were lysed in 200 ml of 1´ passive lysis buffer (Promega) on a shaking platform for 15 min at room temperature. The cell lysates were centrifuged for 5 min at 16,000 ´ g at 4°C to remove cell debris. A total of 20 ml of cleared supernatants was assayed for RLUC activity by the addition of 1 mg of substrate coelenterazine in 100 ml of 0.05 M sodium phosphate buffer (pH 7.0) followed by photon counting in the luminometer (model T 20/20; Turner Designs, Sunnyvale, CA) for 10s. RLUC activities were normalized by measuring the protein concentration by Bradford assay (Bio-Rad, Hercules, CA) and the FLUC activity was normalized by luminometer assay using 100 ml of the LARII substrate from Promega. RLUC activity was expressed as normalized relative light units per microgram of protein.Western Blot Analyses of Endogenous ERa and Intramolecular Folding Sensor
. To study the impact of different ligands on the protein expression of cellular ERa, the MCF-7 cells were exposed to different ligands (1 mM 17b-estradiol, diethylstilbestrol, genistein, tamoxifen, or raloxifene) or carrier control (1 ml/ml DMSO) for 18 h. Cells were collected by trypsinization and lysed in PBS by sonication. The lysed cells were centrifuged at 16,000 ´ g to remove cell debris, and protein content was determined by Bradford assay (Bio-Rad). A total of 10 mg of the total protein was resolved by 4-12% gradient SDS/PAGE (Invitrogen) and electroblotted to a 0.2-mm nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked with TBS (pH 7.6) with 0.01% Tween 20 (TBST buffer) containing 5% nonfat milk for 3 h. The membrane was further incubated in the same buffer containing rabbit polyclonal anti-human ERa antibody overnight at 4°C with on a rotating platform. The membrane was washed with TBST three times and incubated with HRP-conjugated goat anti-mouse antibody for 2 h at room temperature. The membrane was washed three times with TBST buffer before incubation with the chemiluminescent HRP substrate LuminoGlo (Cell Signaling, Beverly, MA) following the manufacturer's instructions. The same membrane was stripped and reprobed with mouse anti-human b-actin or mouse anti-human a-tubulin (Sigma) to control for protein loading. Similarly, to study the impact of different ER ligands on expression of intramolecular folding sensor, the 293T cells transiently transfected with pcDNA-N-rluc-hER281-549-C-rluc were exposed to 17b-estradiol, diethylstilbestrol, genistein, tamoxifen, and raloxifene (1 mM) for 18 h. Total proteins from the cell lysates were used for Western blot analysis using a mouse monoclonal anti-RLUC antibody (Chemicon, Temecula, CA) as described above.Optical CCD Imaging of ER Ligand-Induced Intramolecular Folding in Living Mice.
All animal handling was performed in accordance with Stanford University Animal Research Committee guidelines. For imaging in living nude mice (nu/nu), 293T cells stably expressing fusion proteins N-RLUC-hER281-549-C-RLUC and N-RLUC-hER281-549/G521T-C-RLUC were used. Mice were anesthetized by i.p. injection of »40 ml of a ketamine and xylazine (4:1) solution, and five million stable 293T cells expressing wild-type and mutant sensor were implanted on either side of the animals' hind limbs. The animals were imaged immediately and 18 h after i.p. injection of the 0.5 mg of the ER antagonist raloxifene. To image RLUC activity, 100 ml of coelenterazine (50 mg) in PBS was injected via tail vein 5 sec before imaging. All mice (n = 3) were imaged by using a cooled CCD camera (Xenogen IVIS; Xenogen, Alameda, CA), and photons emitted from the mice were collected and integrated for a period of 5 min. Images were analyzed by using Living Image software (Xenogen) and IGOR image analysis software (Wavemetric, Lake Oswego, OR). To quantify the number of emitted photons, regions of interest were drawn over the area of the implanted cells, and the maximum photons per second per square centimeter per steradian were obtained as previously described (3, 4).1. Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I, Chambon P (1989) EMBO J 8:1981-1986.
2. Paulmurugan R, Massoud,TF, Huang J, Gambhir SS (2004) Cancer Res 64:2113-2119.
3. Bhaumik S, Gambhir SS (2002) Proc Natl Acad Sci USA 99:377-382.
4. Paulmurugan R, Umezawa Y, Gambhir SS (2002) Proc Natl Acad Sci USA 99:15608-15613.