Firefly Protein Lets Researchers Monitor Molecule Linked To Cancer

Thetarget molecule, known as IKK (for IKappa kinase), regulates processesthat can trigger dramatic changes in cellular physiology. Scientistshave linked these changes to many different disorders.

"Our newsystem allows researchers to monitor whether drugs for these conditionsare hitting this exact molecular target in cell culture and laboratoryanimals," says senior investigator David Piwnica-Worms, M.D., Ph.D.,professor of molecular biology and pharmacology and of radiology.

Piwnica-Wormsand lead author Shimon Gross, Ph.D., a postdoctoral fellow, measuredlight from the firefly protein, luciferase, to monitor IKK activity intumor cells and inflamed liver cells in live mice. They also showedthat the technique can greatly reduce the costs of tests that establishthe best dosages for drugs that target IKK. Their results appear in theAugust 2005 issue of Nature Methods.

IKK stands at a pivot pointin the middle of an important set of linked chain reactions known asthe NF-KappaB pathway. The pathway can start at many differentreceptors on cell surfaces; its finish changes the activity levels ofvarying genes. The result, according to Piwnica-Worms, is that thepotential reaction patterns in the NF-KappaB pathway form anhourglass-like shape, fanning out among many options at the start,narrowing in the middle, and again fanning out among many options atthe end.

"At the waist of that hourglass is IKK," he explains."This appears to put it in a position to be the key regulator of thepathway, and that has made it a subject of great interest both from theperspective of understanding how this pathway works and from that ofdeveloping new drugs for conditions that involve this pathway. "

Piwnica-Worms'laboratory has previously developed techniques that use luciferase tomonitor protein-protein interactions. Researchers can employ aninstrument known as an in-vivo bioluminescence camera to take real-timemeasurements of light from luciferase in cell cultures and in cellswithin live animals.

To use the firefly protein to monitor IKK,Gross altered cell lines to genetically fuse the luciferase protein toIKB (IKappaB), the protein that comes immediately after IKK in theNF-KappaB pathway. When the pathway is enabled, IKK triggers reactionsthat lead to the degradation of IKB. In cells with genetically alteredIKB, the attached luciferase is broken down too, meaning scientists candetect increased IKK activity via decreased light from the cells.

"Thisis like doing in-vivo pharmacodynamics and pharmacokinetics," saysPiwnica-Worms in reference to the sciences that study the effects,distribution and dissipation of drugs. "Traditionally the only ways wecould do those kinds of studies were either to test for levels of thedrug in the blood or to label the drug with a radioactive tracer.

"Inthe case of NF-KappaB, there were also methods that monitored IKKactivity via changes in the levels of gene activation at the end of thepathway," he notes. "But those took hours to days to deliver results,and our approach works continuously and in real time."

In theirstudy, Gross and Piwnica-Worms tested the technique in live mice bytransplanting genetically altered tumor cells and by using a techniquethat inserted the fused IKB/luciferase protein into liver cells only.They are currently working to develop a line of mice with theIKB/luciferase fusion built into its genetic code.

In addition,they showed that the system is not only helpful for learning if a drugis having the desired effect, it can also be used to fine-tune drugdosage for maximum benefit.

"One of the reviewers of our papersuggested that we should use the system to produce a full dose-responsecurve, which helps establish how to best use a drug," Piwnica-Wormssays. "Establishing that normally takes 6 months and 300 mice. With ourmonitoring technique, Shimon did it in a 5-day period using 30 mice.That's going to lead to tremendous cost savings."

Because theluciferase-based monitoring system allows monitoring in live animals,Gross could perform multiple tests on the same mouse over time. He wasalso able to monitor the mice for individual variances that couldinappropriately bias the results.

Gross S, Piwnica-Worms D.Real-time imaging of ligand-induced IKK activation in intact cells andin living mice. Nature Methods, August 2005.

Funding from the National Institutes of Health.