Fireflies aren't flies; they're beetles. Nor do these ubiquitous Lampyridae insects (of which there are 2,000 species worldwide) emit fire when they brighten the summer night with their winking, blinking pinpoints of nearly heatless light. These bioluminescent mating signals cast bluish-green flashes or glows of luciferase reporter or marker molecules - much used by researchers to flag biological materials, mainly genes and their proteins.

Now those misnamed fireflies are shedding light on how to test new therapeutic drugs and treat diseases. Their versatile technique is reported in the Early Edition of PNAS, the Proceedings of the National Academy of Sciences, released online Dec. 10, 2002. The article is titled: "Noninvasive real-time imaging of apoptosis." Its co-senior authors are imaging radiologist Brian Ross and radiation oncologist Alnawaz Rehemtulla, both at the University of Michigan in Ann Arbor.

"We have inserted the gene for a firefly's glow-producing luciferase molecule into mice with cancer," Ross told BioWorld Today, "and kept it from shining its telltale beacon of light until the malignant cells started to die in response to the cancer treatment. The process that makes fireflies glow and twinkle at night can also shed light on how new therapeutic drugs are working."

To which co-author Rehemtulla added, "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. It could be used to speed up the testing of new drugs for cancer, ischemic stroke, AIDS, autoimmune disorders, blood diseases, heart-attack damage, neurodegenerative diseases and other disorders where drugs are needed to either kill cells or stop cell death."

Luciferase, the enzyme that lights up the snouts of light-emitting, bioluminescent beetles, has been used for years in biomedical research - but with the glow always turned "on" to create a constant stream of light. The Michigan co-authors report they have discovered a way to switch the bioluminescence "off" until an apoptotic drug causes a cell to start dying.

Estrogen Gets Into The Construct's Act

"We did this," Ross related, "by attaching luciferase to another protein, a portion of the receptor for the hormone estrogen. Its receptor, ER, is known to squelch the action of attached proteins, such as luciferase's light-releasing chemical reaction. To this we added another switch that could turn the luciferase on, but only when the cell was in the self-destructive process of apoptosis.

"We developed this light switch,' Ross continued, "by inserting a tiny section of protein between the luciferase and the estrogen receptor protein, at a site called DEVD. A well-known enzyme, caspase-3, is the key agent involved in the apoptotic process. It's most active when cells are dying. If a cell-killing drug was working successfully, caspase-3 would cleave the DEVD site on the luciferase-ER complex, thus allowing light to leak out."

The team tested its on-again, off-again construct on transgenic mice in which tumor cells were implanted, continually expressing the reporter molecule. "We made a human glioma [brain cancer] cell line that constitutively expressed its luciferase reporter molecule with the estrogen receptors attached on both ends of it. So the genetic information was being produced within the cell. The cell's genetic machinery expressed that molecule in the cytoplasm where it is residing.

"We implanted that glioma cell line with its built-in construct in nude mice," Ross continued, "by subcutaneous injection. It then grew into a solid tumor mass. We treated this with a therapeutic molecule called TRAIL, which induced apoptosis. TRAIL," Ross explained, "is a tumor necrosis factor, a small peptide that binds to a cell-death receptor on the surface of the cell, and initiates the signal cascade that induces apoptosis.

"The construct as a whole is a piece of DNA that we made to generate a specific reporter probe. It's chimeric molecule consists of a double-ER and luciferase combination, which when expressed in a cell won't produce much light. But when apoptosis signaling is turned on, the visible light activity, or photon emission, brightens up seven- to eightfold.

"DEVD consists of four amino acids that are recognized specifically by caspase-3," Ross recounted. "When the ER is bound to the luciferase molecule, it stops that enzyme from glowing. Then the ER must be released from the luciferase to reactivate it. That caspase, which is a protease, cleaves only to a protein that has those four-amino-acid sequences. So that's what gives us the specificity. When that's there on either side, the scissors come in, clip off both ends, and free the ERs from the luciferase. That stops the quenching of it, and reactivates the light output.

"The beauty of the construct is that it's always there," Ross observed, "because it's built into the genetic machinery of the glioma cells. We put it in before we put them into the mice. That's what's super-special about this technique. It's a molecular approach to detection of apoptosis noninvasively. You're not injecting a molecule that goes in and binds to something and then starts glowing, or anything like that. The genetic message goes into the machinery of the cells, so when you make a transgenic animal, you can put this in the neurons. So all of the neurons in the brain will express this reporter. Now if you make the animal model ischemic stroke, stroke injury can be mediated by caspase-3 activation. Thus, neuronal death can be detected by this reporter protein if it's a caspase-3-mediated scenario. And you could put it in the glial cells, the kidneys, the liver, wherever you want to insert it, in a transgenic animal. So you give your mouse the imaging reporter of choice."

From Patent Filing To License To Sublicenses

"As for the experiment's results," Ross said, "we showed that we could noninvasively detect this whole caspase-3 activation process in the living animal. And we validated it by very careful tissue studies afterward.

"The university has applied to patent our technique," Ross noted, "and licensed it to Molecular Therapeutics Inc., also in Ann Arbor. That firm has sublicensed it to other companies for use in the drug discovery process. Rehemtulla and I are principal co-inventors and," Ross concluded, "we both hold key positions at Molecular Therapeutics."