A team of researchers at Swiss pharma giant Novartis has developed a high-content imaging-based assay to study protein prenylation based on a green fluorescent protein reporter tagged with the prenylation motif of human H-Ras.
According to the researchers, the assay can distinguish real inhibitors of the mevalonate pathway from toxic compounds because cytosolic and/or nuclear localization of the GFP-CaaX reporter protein can be observed in cells with normal morphology. Only later, or with higher compound concentrations, is apoptosis or cell shrinkage detectable, the researchers said.
The Novartis team has developed a human osteosarcoma cell line that stably expresses a GFP-CaaX reporter protein, where the “C” in the motif refers to cysteine, the “a” refers to an aliphatic amino acid, and the “X” is typically a menthionine, serine, alanine, or glutamine in proteins being farnesylated, and leucine in proteins being geranylgeranylated.
The cells express GFP with a C-terminal tail that gets prenylated and palmitoylated. These lipids target GFP to the plasma membrane.
Inhibitors of prenylation prevent this lipid modification, and thereby also prevent targeting of the GFP to the plasma membrane. After incubating for between 24 and 48 hours in the presence of such inhibitors, the newly synthesized, non-modified GFP accumulates in the cells, and the prenylated GFP gets degraded by the normal protein turnover. Localization of GFP in cells can be imaged with any cell imager, and the images can be analyzed with different software programs.
After nine days of selection with G418, the GFP-expressing osteosarcoma cells were enriched by fluorescence-activated cell sorting using the BD FACSCalibur. Cellular images were acquired using the IN Cell Analyzer 3000 from GE, and localization of the GFP-CaaX motif was analyzed using the Plasma Membrane Trafficking module of the IN Cell Analyzer using the D peak as a readout.
The authors validated their assay using several inhibitors of the mevalonate pathway and siRNA against farnesyl pyrophosphate synthase. To quantify the RNA, the researchers used Agilent Technologies’ RNA 6000 Nano LabChip kit. They performed quantitative PCR using the ABI Prism 7000 sequence-detection system from Applied Biosystems.
Until recently, cell-based assays to study protein prenylation have been tedious and low-throughput. “I think most people know what it means to analyze several samples with Western blotting: You need relatively large amounts of cells, and you need to prepare a minimum of a whole cell lysate,” Marjo Simonen, lab head of discovery technologies at Novartis, told CBA News this week in an e-mail.
She said that for some applications, a researcher must fractionate the cells, run the gels, blot them, treat the blots with antibodies, and in the end, use some method for visualization of the bands.
In addition, the intensity of the bands must be quantified with a scanner. “This requires several steps, is slow and semi-quantitative, and limits the number of samples that can be analyzed per gel,” Simonen said.
The mevalonolactone assay requires a change of medium and a centrifugation step to separate the membrane fraction from the rest. These are not yet automated processes. In addition, radioactive chemicals are required, which, as Simonen put it, “we want to avoid whenever possible.”
The luciferase reporter assay was not yet published when the investigators at Novartis started developing their imaging assay, said Simonen. Once it was published, they did not have access to the appropriate cell line, and generating a similar cell line would have taken a relatively long time. Moreover, Simonen said, “We prefer the imaging assay, since seeing the cells provides so much more information.”
The use of this assay in primary screening for propoptotic inhibitors would require more sophisticated image-analysis tools, the investigators said. Simonen said her team plans to use other image-analysis software to distinguish the truly active compounds from the “toxic only” compounds. “This should be doable with the Developer software from GE,” she said.
Simonen said that Novartis has no plans to commercialize the assay, but “if someone wants to do it, he or she should feel free to contact us.”