Dec. 18, 2002 ANN ARBOR, MI – The process that makes fireflies glow bright in the summer night can also shed light on how well new medicines work, showing immediately whether the drugs are effective at killing cells or causing other effects.
That's the conclusion of a team of scientists from the University of Michigan Health System, who report that they have inserted the gene for a firefly's glow-producing molecule into mice with cancer, and kept it from producing its telltale beacon of light until the cells started to die in response to cancer treatment.
Using a highly sensitive camera, the researchers were able to immediately detect the faintest traces of the firefly light as it passed outside the bodies of the mice.
The findings, published in the Dec. 24 issue of the Proceedings of the National Academy of Sciences and posted online last week, promises to give researchers a new way to get real-time information on whether new medicines are working.
It could be used to speed up the testing of new drugs for cancer, stroke, AIDS, auto-immune disorders, blood diseases, heart attack damage, nerve-degenerative diseases like Alzheimer's disease, and other disorders where drugs are needed to kill cells, or to stop cell death. It could also be used to monitor other cell processes.
"This is the first time anyone has been able to make real-time images of apoptosis -- the process of cell death that is so important to so many diseases and treatments," says lead author Alnawaz Rehemtulla, Ph.D., associate professor of radiation oncology at the U-M Medical School and co-director of the U-M Center for Molecular Imaging. "This proves that we can see what's going on at the molecular level while the drugs are working, giving results in days or weeks instead of months or years.'
The process is made possible through the use of luciferase, the enzyme that triggers a chemical process that lights up the tails of fireflies. The light-emitting process, called bioluminescence, has been used for several years in biomedical research -- but with the glow always turned "on" to produce a constant stream of light.
The U-M team, led by Rehemtulla and Brian D. Ross, Ph.D., professor of radiology and co-director of the center, reports they have discovered a way to turn the light "off" until a drug causes a cell to start dying.
They did this by attaching luciferase to another protein, a portion of the receptor for the hormone estrogen. The estrogen receptor, or ER, is known to squelch the action of attached proteins, such as luciferase's light-releasing chemical reaction.
Then, they added a "switch" that could turn the luciferase on -- but only when the cell was in the self-destruction process called apoptosis.
The ability to make images of apoptosis in real time could help pharmaceutical companies and academic researchers screen new drugs rapidly, for all types of diseases that are characterized by over-growing cells – or cells that die too soon.
For example, scientists are studying drugs that can keep brain cells from dying after a stroke, a process that occurs through apoptosis and can cause permanent disability. Conversely, cancers and blood disorders that stem from over production of certain cell types are often treated with drugs aimed at killing extra cells.
But the U-M team believes its technique could also help with drug discovery and testing in diseases where other molecular mechanisms besides apoptosis are involved.
The team's discovery hinges on the ability to turn the luciferase glow off until a cell process occurs. They developed this "light switch" by inserting a tiny section of protein between the luciferase and the estrogen receptor (ER) protein. Called DEVD, the section is known to be targeted for cutting by an enzyme called caspase-3 -- the key agent that sets off apoptosis. Because caspase-3 is most active when cells are dying, the team felt that it would cut the DEVD site on their luciferase-ER complex, allowing light to escape, if a cell-killing drug was working.
They were right. In the new report, the team shows that the luciferase-DEVD-ER complex released much less light than luciferase alone in either cell cultures or in mice with cancer that had been injected with a DNA sequence encoding the complex. When they added another ER and DEVD to the other side of the luciferase, the amount of light emitted was cut even further.
But when the researchers added an experimental cancer-fighting drug called TRAIL, which is known to cause apoptosis, it was just like turning on a light switch.
As the drug caused the activation of the caspase-3 enzyme, the bonds between the luciferase and its light-hiding companion ER molecules were cut at the DEVD cleavage site, and the light-emitting process began. One hour after TRAIL treatment, the amount of light emitted from the mice had gone up by 186 percent. Mice that received a placebo instead of TRAIL emitted only about 21 percent more light than they had before.
Even though only a few photons, or particles of light, can escape a mouse body from the glowing luciferase, the researchers were able to detect them with a bioluminescence imaging camera that can detect single photons.
The camera, which is kept in a light-proof chamber at ultra-cold temperatures to keep out all stray energy, produced vivid images showing the intensity of light -- and therefore the rate of cell death -- in different regions of the cell cultures or mouse cancer tumors. Rehemtullah notes that the technology is sensitive enough to pick up cell death in areas as small as 100 cells.
"The enzyme was turned on in a protease-dependent manner, and though we used caspase-3 in this instance, we believe it could be any protease that's important to any cellular process," Ross emphasizes. "And, we believe other reporters could be used, including radioactive positron-emitting molecules that can be imaged using PET scanning." He noted that preliminary work on these approaches, and efforts to develop a transgenic strain of mice containing the genes for the luciferase complex, is already under way at UMHS.
The research was funded by the National institutes of Health through grants supporting the Center for Molecular Imaging, and by the Michigan Life Sciences Corridor.
The U-M has applied for a patent on the technique, and licensed it to a local biotechnology company, Molecular Therapeutics, Inc. Both Rehemtulla and Ross hold key positions at Molecular Therapeutics (http://www.moleculartherapeutics.com).
Other authors are former postdoctoral fellow Bharathi Laxman, Ph.D., research associate Daniel Hall, research fellow Mahaveer Swaroop Bhojani, radiation oncology resident Daniel Hamstra, and radiology professor Thomas Chenevert, Ph.D.
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