Researchers at Washington University School of Medicine in St. Louis have imaged the activity of an important component of the cell’s garbage-disposal system in living cells and in whole animals. Defects in the component, known as a proteasome, are associated with cancer, neurodegenerative diseases and other disorders.
Researchers at Washington University School of Medicine in St. Louis have imaged the activity of an important component of the cell’s garbage-disposal system in living cells and in whole animals. Defects in the component, known as a proteasome, are associated with cancer, neurodegenerative diseases and other disorders. Using this imaging method, the investigators found that an experimental anticancer drug that inhibits proteasome function does in fact reach effective levels in experimental tumors, and that the drug may become more potent after weeks of use. “The study demonstrates the value of molecular imaging techniques in general for assessing an experimental drug’s activity,” says first author Gary D. Luker, M.D., assistant professor of radiology. “Most importantly, it enables us to track changes in the same mouse over several hours or days to see how drug effects change with time and vary within and among mice.” Typically, scientists estimate proteasome activity indirectly by testing white blood cells. But bioluminescent molecular imaging may speed drug development by providing a more effective way to study an experimental drug’s effects on tissues and its metabolism and distribution in the body. It also may help estimate safe and effective dosage levels prior to human testing. “A drug’s activity in white cells in the bloodstream may not reflect its activity in a tumor, the brain or other target tissue,” says principal investigator David R. Piwnica-Worms, M.D., Ph.D., professor of radiology and of molecular biology and pharmacology. “Bioluminescence imaging allows us to measure proteasome activity within the target tissue.” Proteasomes dispose of many important proteins, including those that regulate cell division, those made by viruses and those that are faulty or malformed. Cells first tag proteins destined for destruction with a small molecule known as ubiquitin. Proteins labeled with ubiquitin are then transported to a proteasome for degradation. Piwnica-Worms, Luker and colleagues began their study by adding a gene for luciferase, a protein made by fireflies, to a line of laboratory-grown cancer cells known as HeLa cells. The cells thereafter churned out a steady stream of luciferase. They then added luciferin, a compound also produced by fireflies that emits light when combined with luciferase, causing the cells to glow. They recorded this bioluminescence using a charged-coupled device, or CCD camera. Next, the investigators fused a luciferase gene and a ubiquitin gene. They added this so-called fusion gene to a second group of HeLa cells, causing the cells to produce luciferase tagged with ubiquitin. Thus, the luciferase made by these cells was destroyed by proteasomes almost as fast as it was made. These cells therefore glowed only weakly when exposed to luciferin. The researchers then added a proteasome inhibitor to the luciferase-ubiquitin-producing cells. The inhibitor, an experimental anticancer drug known as bortezomib (or Velcade,‰ formerly known as PS-341), caused the cells to glow more brightly. “This indicated that the drug had slowed the destruction of the luciferase-ubiquitin protein, presumably by blocking the action of proteasomes,” says Piwnica-Worms. Next, the researchers grafted both groups of HeLa cells onto immune-deficient mice, which then developed tumors. Tumors with the luciferase-only gene glowed brightly as measured by the CCD camera, while tumors with the luciferase-ubiquitin gene glowed weakly. The mice then were given either a low, medium or high dose of bortezomib. The investigators found that bioluminescence in tumors with the luciferase-ubiquitin gene increased with the drug dose. The highest and most effective dose (1.0 microgram/gram) of drug blocked proteasome activity by almost 80 percent. Luminescence greatly increased within 30 minutes, peaked at 6 hours and returned to baseline within 46 hours. After two weeks of treatment, luminescence in these tumors no longer rose and fell with each dose but remained consistently high. “This finding suggests that the drug’s chemistry changes in the body with time,” Piwnica-Worms says. “It also shows the power of molecular imaging to provide information that might otherwise be missed by blood tests and other less direct measures of drug activity.” Next, the investigators will apply bioluminescent molecular imaging to studying drug combinations in cell and animal models. |
