Evolutionary Shocker?

In the late 1970s, a television series based on the popular comic book The Incredible Hulk became a surprise hit. The comic related the adventures of a scientist who studied the effect of radiation on physical strength. After a freak accident in a nuclear reactor, the researcher would temporarily transform into a gigantic, green monster with incredible strength–the Hulk–whenever he experienced stress. The clothes-ripping, skin-coloring transformation of the scrawny scientist into the Hulk was an amusing staple of every episode of the television show.

Such a stress-induced makeover is, of course, impossible. Or is it? Two provocative studies, one published in 1998 on fruit flies and one this year on plants, suggest that organisms developing under stressful conditions can unleash novel mutant forms.

In doing so, some biologists speculate, some offspring may quickly adapt to new environmental conditions and improve their species’ odds of survival.

This isn’t comic book science. The studies have thrown the spotlight on a molecule called heat shock protein 90 (Hsp90) that’s central to the stress-induced effects. Susan Lindquist of the Whitehead Institute of Biomedical Research in Cambridge, Mass., who coauthored the two studies, contends that when Hsp90 activity is disrupted during development, genetic variation that’s normally concealed can emerge and generate diverse physical forms of a species.

Changes in Hsp90 activity “can act as a mechanism for evolutionary change by covering and uncovering genetic variation,” says Lindquist.

Evolutionary plant biologist David Baum of the University of Wisconsin–Madison suspects that Hsp90 doesn’t stand alone in this surprising story. He says the Hsp90 findings are “profoundly important” because they show that hidden genetic diversity exists within species and can erupt when conditions change.

Important clients

In 1962, scientists studying the fruit fly Drosophila melanogaster came across a family of molecules quickly dubbed heat shock proteins. The researchers discovered that when flies are exposed to brief periods of elevated temperatures–a heat shock–their cells activate a group of specific genes. Biologists found that the products of these genes, the heat shock proteins, help newborn proteins fold into their proper shape and become stable. Otherwise, at the higher temperature, many new proteins are unstable or don’t function.

The molecular helpers, also called chaperones by some scientists, battle more than heat. Cells make heat shock proteins when flies experience a variety of stresses, such as radiation, starvation, and dehydration. And heat shock proteins aren’t limited to flies. Seemingly all plants, animals, and even microbes produce these molecules when cells are stressed.

For more than a decade, beginning when she was at the University of Chicago, Lindquist has been studying Hsp90. Unusual among the heat shock proteins, Hsp90 is abundant in cells all the time, not just during times of stress. Working with yeast cells, Lindquist and her colleagues showed that Hsp90 has a select number of other proteins, its clients, that it stabilizes and folds under normal conditions. Some of these Hsp90-dependent proteins have crucial roles in signaling between cells during an organism’s development. Other clients are proteins important to cell growth.

Curious about Hsp90’s role in an organism more complex than a yeast cell, Lindquist and her University of Chicago colleague Suzanne Rutherford turned to fruit flies. In flies, if both copies of a fertilized egg’s Hsp90 gene are defective, the insect doesn’t develop at all. But if one copy works and the other is mutated, causing Hsp90 activity to be 50 percent of normal, the egg usually develops into a fertile adult.

When Rutherford and Lindquist studied flies with such a half-portion of Hsp90, however, they observed physical abnormalities in about 5 percent of the insects. Misshapen or missing eyes, stubby wings, deformed legs, or antennae growing in the wrong places are just some of the oddities that the researchers documented. Similar developmental defects emerged in the offspring of flies raised on food containing the Hsp90 inhibitor geldanamycin, a compound now attracting the interest of cancer researchers (see “Hot New Cancer Therapy,” below).

The two biologists considered several explanations for their observations. In the scenario they ultimately favored, Hsp90’s role as a chaperone normally suppresses the disruptive influence of mutations in genes crucial to an animal’s development. Hsp90 could do this by folding slightly mutated versions of its clients into their normal shapes, for example.

By breeding the Hsp90-deficient flies, the researchers showed that individual abnormalities are inherited consistently from generation to generation. That, they said, supports the theory that the defects are caused by mutations freed of some Hsp90 control, rather than by a random mishap triggered by reduced Hsp90 activity at some point during a fly’s development.

The role of Hsp90 as a heat shock protein is central to the researchers’ conclusions. They argue that if cells are stressed during development, Hsp90 becomes diverted from its normal protein clientele to stabilize other proteins in cells. As a result, developmental signals normally kept in line by Hsp90 miss that influence, go awry, and change an organism’s body plan.

For evolution’s sake, this isn’t necessarily a bad thing. With Hsp90 keeping a mutated gene’s protein under control, an organism could accumulate more genetic variation than it would otherwise. The organism can then expose that variation in response to environmental stress, permitting natural selection to act upon it, says Lindquist.

At the end of their 1998 paper on the fly research, Rutherford and Lindquist offered a provocative conclusion. “We have provided what is, to our knowledge, the first evidence for an explicit molecular mechanism that assists the process of evolutionary change in response to the environment,” they said.

The biologists raised eyebrows even more by suggesting that this Hsp90-based evolution mechanism could help explain the rapid bursts of body-plan changes that occasionally punctuate the fossil record. Most evolutionary theory predicts small, gradual changes in a species over long periods and is hard-pressed to explain such morphological explosions.

Weird weeds

Many biologists remained skeptical of Rutherford and Lindquist’s conclusions. To suggest that Hsp90 helps organisms adapt to their environments was a radical notion, especially given that the mutant fruit flies generated by reduced Hsp90 activity had no obvious advantages over normal flies. If anything, their physical defects would make them less likely to survive in the environment and pass on their genes.

In the June 6 Nature, however, Lindquist and two of her University of Chicago colleagues, Christine Queistch and Todd A. Sangster, add another chapter to the story of Hsp90 and evolution. They report that if seeds of the mustard weed Arabidopsis thaliana grow in soil containing the Hsp90 inhibitor geldanamycin, the resulting plants frequently display altered forms, including some that could in theory help a plant adapt to its environment.

The researchers found that geldanamycin-exposed plants sometimes grew leaves with an altered shape or color, for example. In other cases, plants had hairier roots than normal, which could improve their uptake of nutrients and water maintenance within the plants. “Having a lot of root hair could be advantageous under some circumstances and not under others,” says Lindquist.

As they had in flies, the researchers showed that a trait produced by Hsp90 inhibition could consistently pass from one generation of the mustard plant to the next. This indicated that the trait is encoded in the organism’s genes but expressed only when Hsp90 activity is reduced. But after several generations, notes Lindquist, these traits can persist even when a plant’s Hsp90 production returns to normal. This finding is mirrored by fly results, in which an exposed abnormality can persist into later generations that have normal Hsp90 activity.

The investigators also demonstrated that, within a line of A. thaliana plants, they could alter the same trait either by growing seeds in geldanamycin-laced soil or by growing them at 27C instead of the typical 22C. The warmer temperature presumably shocks the plant cells, diverting Hsp90 from its normal clients to a generalized stress response. In essence, the researchers say, the change in environmental conditions provided a natural means of inhibiting Hsp90’s normal role during the plant’s development.

Finding that Hsp90 may act similarly in a plant and an insect suggests that its role in enabling organisms to stockpile genetic variation arose early in the history of life, concludes Lindquist. “It shows that this ability to hide genetic variation and reveal it is a very, very ancient thing,” she says.

By documenting that Hsp90 inhibition produces plant forms that conceivably have advantages in some environments, the Lindquist team’s new report makes a “considerably stronger case” for Hsp90’s role in evolution, says developmental biologist Bruce P. Brandhorst of Simon Frasier University in Burnaby, British Columbia.

Accidents happen

Lindquist and her colleagues are careful to say that they don’t hold that Hsp90 arose to help organisms evolve. Hsp90’s primary role is to shape crucial cellular proteins, and any influence over evolution is an “accidental” consequence of that function, says Lindquist.

Other investigators question the group’s emphasis on Hsp90. “People are fixated on the heat shock proteins. I’m not convinced that’s the story,” says Baum. He speculates that changing the activity of other proteins during development may also unleash hidden variation in plants and animals.

Evolutionary biologist Günther P. Wagner of Yale University, who has followed the Hsp90 story closely, says that many issues must be resolved before he and his colleagues accept the idea that the protein affects evolution by unleashing genetic variation at times of stress. “Is this mechanism at all invoked under natural conditions?” he asks.

Somehow, says Wagner, scientists must find a way to experimentally test whether drops in Hsp90 activity help members of a species adapt to new circumstances. For example, investigators might examine whether a strain of Hsp90-inhibited flies does better in changing environments than a normal strain does.

Lindquist is skeptical that such a study would be meaningful. Instead, she and her colleagues intend next to investigate whether they can use geldanamycin to create plant variants that are useful to farmers. Hsp90 inhibition may offer a more natural way to create new plants than the controversial practice of genetic modification does, the researchers suggest.

In general, says Lindquist, her group’s work on Hsp90 should persuade evolutionary biologists that they have to focus on more than just genes. “It’s really very important to not just look at DNA but to understand how proteins fold,” she says.

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Hot New Cancer Therapy

Heat shock protein 90 may provide a good target for pharmaceuticals

The endless war that goes on between microbes may have spawned a new weapon for the war on cancer. Heat shock protein 90 (Hsp90), the molecule at the center of controversy concerning its possible role in evolution, sits at the center of this unexpected story, as well.

In the 1970s, scientists isolated a family of natural antibiotics, called ansamycins, that microbes secrete–seemingly, to kill off competitors. In the next decade, as part of its ongoing effort to screen for novel drugs, the National Cancer Institute (NCI) in Bethesda, Md., tested one of these antibiotics, geldanamycin, against a variety of tumor cell lines. The scientists found that the drug has some limited anticancer properties.

Developing the drug wasn’t a high priority, however, because researchers believed that geldanamycin stops cancer cells by inhibiting a family of enzymes called tyrosine kinases. Since normal cells also depend on these enzymes, investigators suspected that geldanamycin would be too toxic.

In 1992, however, NCI’s Leonard Neckers and his colleagues discovered that geldanamycin doesn’t directly inhibit the enzymes. The researchers found instead that the drug binds to a pocket on Hsp90 and disrupts its function. In doing so,

geldanamycin apparently prevents Hsp90 from stabilizing a variety of proteins, including several tyrosine kinases, required for many types of cancer cells to grow and spread.

The discovery of geldanamycin’s target gave Hsp90 investigators the first easy way to inhibit the protein’s activity and study its roles in cells. It also triggered new interest among cancer researchers, who began to test geldanamycin in animals with different cancers. “It proved to have good antitumor activity in certain animal models,” says Neckers.

Geldanamycin had a significant flaw, however. For reasons unrelated to Hsp90, it causes liver damage. Seeking a safer alternative, investigators turned to a geldanamycin derivative called 17-AAG. “It’s tolerated very nicely in animals, much better than the parent compound,” says Neckers. “You can give a lot more to animals without any toxicity.”

About 2 years ago, a few cancer centers funded by NCI began testing 17-AAG in several dozen people. “There have been some minor [antitumor] responses but nothing that would knock your socks off,” says Neckers.

He’s not discouraged, however, because these initial trials were primarily designed to establish the safety of the drug and the best way to administer it. Physicians and NCI are now discussing larger trials of 17-AAG that would target cancers thought particularly susceptible to Hsp90 inhibition.

On the basis of animal studies and knowledge of what proteins Hsp90 binds, breast, prostate, and certain lung cancers are likely to be priorities. There are also data indicating that the drug may help people with leukemia and other cancers resistant to the recently approved and promising drug Gleevec, notes Neal Rosen of Memorial Sloan-Kettering Cancer Center.

Adding 17-AAG to current chemotherapy regimens could also prove fruitful, according to researchers. “It potentiates the toxicity of other agents,” says Neckers. “It has activity on its own, but it has even better activity in combination with [cancer] drugs like taxol.”

Rosen is so optimistic about the potential of therapy that targets Hsp90 that he helped found a San Diego-based company called Conforma to develop better inhibitors of the protein.

“Things have come along nicely in the last 10 years,” says Neckers.

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