Cellular Pump Sabotages Cancer Drug Studies That Use Glow Chemical
December 13, 2007
Johns Hopkins scientists have by chance discovered that a widely used means of illuminating cancer cells could undermine studies of the potential value of experimental anti-cancer drugs because the natural “pump” that cells use to clear out the chemical light source alters their chemistry.
“Researchers who use markers involving luciferase may want to double-check their findings,” says Martin Pomper, M.D., Ph.D., associate professor of radiology, oncology, and pharmacology and molecular sciences at Johns Hopkins.
Scientists have increased their use of “glowing” markers to track cellular activity in rats and mice, in part, because the tactic is non-invasive and more humane for the animals.
To make the cells glow, scientists use a gene encoding luciferase, an enzyme that causes a chemical reaction responsible for the tiny glow in fireflies. Researchers transfer the luciferase gene into the genetic machinery of cancer cells, which then are injected into an animal, and the enzyme’s glow signals the response of a cell to an anticancer drug.
But in a chance discovery, Pomper, collaborating with John Laterra, M.D., Ph.D., professor of neurology, neuroscience and oncology at Johns Hopkins and the Kennedy Krieger Institute, found that a cellular “pump” that automatically rids cancer cells of its glowing contents over time, can distort test results. They stumbled on the saboteur pump during experiments with a bioluminescent marker in mice that is designed to test the effect of an experimental drug on a cancer-causing gene pathway, called hedgehog.
Pomper’s work focused on an anti-hedgehog compound called HhAntag-691 that his team hoped would turn off the pathway with a single dose. The way the study was designed, his team first bioilluminated the cancer cells with the luciferase gene, then introduced HhAntag-691 into the animals. If it worked, researchers would see no glow in the cancer cells because the hedgehog pathway would be switched off, failing to activate other components in the hedgehog pathway that turn on luciferase.
“But when we added the anti-hedgehog compound, the cells glowed brighter rather than getting dimmer,” says Pomper, whose team from the Johns Hopkins Kimmel Cancer Center and Russell H. Morgan Department of Radiology observed a threefold increase in glow output. The glow is measured by means of a photograph taken through the animal’s skin with a camera that detects bioluminescent wavelengths. “We thought this was bizarre and repeated the experiment many times.”
In an effort to figure out their problem, the researchers repeated the experiment in a cell extract, a replica of the cell environment without the intact cell itself. In this test, the glow dipped threefold. “This suggested that natural pumps on the surfaces of intact cancer cells pump out luciferin, which reacts with intracellular luciferase to cause the cells to glow,” said Pomper, whose report on the subject appeared in the October 1 issue of Cancer Research.
Pomper’s team identified the chemical pump, called ABCG2/BCRP, which is one of nearly 50 of its type. And they warn drug developers to be wary of it.
“If you want to use bioluminescence to test a drug’s action, make sure the cells don’t have this pump,” says Pomper.
The trouble is that many cancers express this pump, and shutting it off would complicate the test, according to Pomper. Still, the discovery has tipped off the researchers to a new target and a test to find inhibitors of it.
“We’re now looking for inhibitors of the pump, which when coupled with standard chemotherapy, could lead to less drug resistance than currently seen for most tumors,” says Pomper.
Funding for the research was provided by the National Institutes of Health and the Dana Foundation.
Other participants in the research are Yimao Zhang, Joseph P. Bressler, Jeff Neal, Bachchu Lal, and Hyo-Eun C. Bhang of Johns Hopkins.
Johns Hopkins Kimmel Cancer Center
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Media Contact: Vanessa Wasta
December 13, 2007