( Stable repor...)Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion,,,Transactivators

Stable reporter cell lines for signal transduction pathway readout

Lisa Hexdall Li Xu Alan Greener Mary Buchanan John Bauer Chao-Feng Zheng
Stratagene

Stratagene now offers the PathDetect HLR-CREB, HLR-Elk1, and HLR-cJun, cell lines ^F for studying signal transduction pathways that converge at the transcription factors CREB, Elk1, and c-Jun. These stable HeLa cell lines feature constitutive expression of the GAL4-CREB, GAL4-Elk1, or GAL4-cJun fusion proteins, respectively. They also contain the firefly luciferase gene, which is controlled by a promoter that responds to GAL4 fusions, as does the HLR cell line that contains only the GAL4-responsive luciferase expression plasmid. When activated by phosphorylation, these fusion proteins bind to the promoter and induce luciferase expression. Therefore, luciferase activity in stable cell lines reflects the activation status of the fusion transactivator and, hence, the activation status of corresponding signaling pathways. The effects of extracellular stimuli on signaling pathways that converge at these activators can be studied directly, without transfection. The potential applications of these cell lines include studying signal transduction and transcription mechanisms and assessing protein-protein interactions in mammalian cells. In addition, these cell lines can potentially be adapted for high-throughput drug screening.

Regulation of gene transcription by extracellular signals is pivotal for controlling growth and differentiation. Because the transcription machinery of eukaryotic cells is located inside the nucleus, signals outside the cells need to be transmitted across the cytoplasmic and nuclear membranes as well as the cytoplasm. This process of signal transmission, better known as signal transduction, involves elaborate networks of signaling molecules (e.g., receptors, protein kinases, and phosphatases) organized in linear or parallel pathways.^1,2 These signaling pathways receive diverse upstream signals that cause distinct downstream changes. Among the better-characterized signal transduction pathways are the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) pathways. When activated by upstream signals, PKA or MAPK translocates into the nucleus and activates transcription factors, such as CREB and Elk1.³ Thus, the activation level of the transcription factors reflects the status of the respective kinases and upstream signaling molecules along the pathway.

To facilitate studies on the in vivo functions of gene products or extracellular stimuli, Stratagene developed the PathDetect trans-reporting systems using GAL4 fusion transactivators as pathway-specific sensors.^4,5,6,7 In these systems, a fusion trans-activator plasmid that consists of the DNA binding domain of the yeast GAL4 (residues 1 to 147) protein⁸ and the activation domain of CREB, Elk1, or c-Jun is used to probe the activity of PKA, MAPK, and JNK, respectively.^4-7,9,10 These fusion trans-activators bind to the promoter in the pFR-Luc reporter plasmid, which contains five direct repeats of the GAL4-binding element and controls luciferase expression.⁴ Either direct or indirect phosphorylation of the activation domain of a fusion trans-activator protein by an uncharacterized gene product activates transcription of the luciferase gene. Thus, expression levels of luciferase reflect the activation status of the respective kinases that define the signal transduction pathways.

To offer expanded capability for studying signal transduction and improved performance for adaptation to high-throughput applications, Stratagene has developed stable HeLa reporter cell lines that express the GAL4-CREB, GAL4-Elk1, or GAL4-cJun. In the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines, luciferase activity reflects the activation status of the chimeric trans-activator protein and, therefore, the respective signaling pathways. By performing simple luciferase assays, researchers can directly evaluate the effects of extracellular stimuli on signaling pathways converging at CREB, Elk1, or c-Jun. Use of these cell lines eliminates transfections, thereby saving time while avoiding the variables that can be caused by fluctuations in transfection efficiencies. The HLR cell line can be used to assess protein-protein interactions in mammalian cells,¹¹ while the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines can be used to study transcription mechanisms. Another application for these cells includes adaptation for high-throughput drug screenings, since many components of signal transduction pathways are potential targets for drug development efforts.

Establishing Stable Reporter Cell Lines

Fig.1

To create stable reporter cell lines with the PathDetect trans-reporting systems, we tried several approaches. Figure 1 outlines our most successful protocol: We first transfected the pFR-Luc plasmid with a hygromycin expression cassette into HeLa cells. Through successive rounds of selection in antibiotic medium, hygromycin-resistant clones were isolated and tested with the PathDetect CREB, Elk1, and c-Jun trans-reporting systems. Cell lysates from these stable clones were then prepared, assayed for luciferase activity, and analyzed. One clonal line, designated as HLR for the HeLa luciferase reporter cell line, was chosen for further experimentation. It gave the highest levels of luciferase activity when tested with the trans-reporting systems mentioned above and had the lowest background of luciferase activity.

Next, in separate experiments, we transfected the HLR cell line with the fusion trans-activator pFA2-CREB, pFA2-Elk1, or pFA2-cJun plasmids. After performing successive rounds of selection with G418, we chose the clones that gave the best response when transfected with the positive control vectors from the PathDetect kits. These clones were then subjected to more extensive analysis.

Testing Stable Cell Lines

Fig.2

Genomic DNA from each stable clone was prepared and used as a template for PCR analysis to confirm that each fusion trans-activator plasmid was stably incorporated into the genomes of the selected clones. To amplify the activation domain for each reporter cell line, we used one primer specific to the GAL4 DNA-binding domain and another primer specific to each of the activation domains. Each primer set successfully amplified a product of the expected size. Analysis of HLR-CREB and HLR-Elk1 cell lines are shown in Figure 2. To further confirm the activity and specificity of these cell lines, we transfected each cell line with plasmids encoding known activators of each pathway. Upon luciferase assay of cell lysates, each cell line showed significant luciferase expression above background (Figure 3).

Fig.3

Additional testing included an analysis of the activation of luciferase activity in these stable cell lines by extracellular stimuli. For example, HLR-Elk1 cells were treated with known MAPK activators, including epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) (Figure 4). Luciferase expression in the HLR-Elk1 cells was five times higher than the control, when cells were treated with 100 ng/ml of EGF, and almost 50-fold higher when cells were challenged with PMA (60 ng/ml). Dulbeccos modified Eagles medium (DMEM) and dimethylsulfoxide (DMSO) were used as negative controls.

Fig.4

For the final testing, we confirmed purity by screening for mycoplasma and established viability after freezing in liquid nitrogen. The HLR, HLR-CREB, HLR-Elk1, and HLR-cJun cell lines are available as frozen stocks containing 5 x 10⁵ cells; each cell line includes the corresponding positive and negative control plasmids.

Conclusions

The HLR-CREB, HLR-Elk1, and HLR-cJun stable cell lines offer a new approach and added convenience for studying the intricate interactions of signal transduction pathways. These cell lines can be used to evaluate whether a new gene product or extracellular stimulus is involved in signaling pathways that converge at CREB, Elk1, and c-Jun transcription factors; furthermore, the cell lines can be used to identify which step along the pathway is targeted. The HLR-CREB, HLR-Elk1, and HLR-cJun cell lines can be adapted for high-throughput screening of inhibitors or activators of these signaling pathways. In addition, the HLR cell line can be used in a variety of other applications, such as assessing protein-protein interactions in GAL4-derived mammalian two-hybrid systems.

REFERENCES

1. Boulikas, T. (1995) Critical Rev. Eukaryotic Gene Exp. 5: 1-77.

2. Karin, M. and Hunter, T. (1995) Curr. Biol. 5: 747-757.

3. Treisman, R. (1996) Curr. Opin. Cell Biol. 8: 205-215.

4. Xu, L., et al. (1997) Strategies 10: 1-3.

5. Xu, L., et al. (1997) Strategies 10: 79-80.

6. Xu, L. and Zheng, C.-F. (1997) Strategies 10: 81-83.

7. Sanchez, T., et al. (1998) Strategies 11: 52-53.

8. Sadowski, I. and Ptashne, M. (1989) Nucl. Acid Res. 17: 7539.

9. Marais R., et al. (1993) Cell 73: 381-393.

10. Smeal, T., et al. (1994) EMBO J. 13: 6006-6010.

11. Dang, C.V., et al. (1991) Mol. Cell. Biol. 11: 945-962.