Cell Suicide

Soon after sperm meets egg, when cells are dividing and dividing again to form the tiny sphere that will grow into an embryo, another—quite opposite—activity begins. Cells die. Specifically, some of the cells inside this little ball commit suicide. Many questions remain about how and why these cells annihilate themselves; however, scientists know that their suicide, termed apoptosis, is essential. If it is prevented, the tiny sphere does not develop.

For life, there must be death.

This yin and yang of cell birth and self-inflicted death repeats itself throughout the human lifespan. Cell suicide helps rid the body of cells that are infected, damaged or simply old and in the way. In fact, every cell in the human body contains the genetic instructions for carrying out its own demise. And under the right circumstances, any cell will.

But scientists took many years to accept the theory of cell suicide and to appreciate that cells conduct their own deliberate and intricately orchestrated murder—a programmed cell death. "It's counterintuitive," admits Marie Hardwick, the David Bodian Professor of Molecular Microbiology and Immunology. Why would a cell kill itself? What good does it do the cell?

Yet Hardwick, PhD, has spent the past 20 years conducting experiments to demonstrate that programmed cell death is as vital to an organism as cell division, and championing theories (hers and others) about when and how cell suicide occurs. Those ideas have not infrequently clashed with scientific dogma, but Hardwick says that challenging convention is essential to good science. "If you don't predict things that are a little out of the box, you don't get anywhere."

Hardwick's office is on the fifth floor of the Bloomberg School. Her window offers a glimpse of the School of Medicine, where she has collaborated with many researchers. Hardwick has also worked closely with scientists at the Bloomberg School, Hopkins' School of Arts and Sciences, and institutions outside Hopkins. She is a basic scientist, who studies the cellular and molecular biology of programmed cell death. Investigators in many different specialties seek her advice and the tools of her lab.

"I think it would be hard to find a disease that does not entail programmed cell death," says Marie Hardwick.

This research cuts across so many scientific disciplines because programmed cell death is a fundamental cell activity, says Hardwick. It is required for proper development; programmed cell death melts away the webbing between a fetus's fingers and between its toes. It destroys cells the body no longer needs; millions, perhaps billions of old cells—skin, liver, lung, intestine and other types—succumb to suicide each day.

Programmed cell death also guards against the slings and arrows of everyday existence. It helps fight off viral infections. It helps prevent cancer—the sloughing of skin following a sunburn being one visible sign of the body ridding itself of potentially pre-cancerous cells. And when apoptosis does not work properly, it can cause disease. Over-zealous cell death contributes to a variety of diseases, such as Alzheimer's disease and AIDS, while cancer involves too little cell death. "In my serious professional opinion, every human disease has the regulation of cell survival at its core," says Hardwick. "I think it would be hard to find a disease that does not entail programmed cell death."

Down the hall from Hardwick's office is her lab where a dozen graduate students and postdocs conduct experiments on cells from mammals, insects and yeast. In one small windowless room, graduate student John Clayton removes a Petri dish of mosquito cells from an incubator and places it on a microscope viewing platform. "Oh, my God. This is so dramatic," he exclaims, peering into the microscope. "Just an hour ago, I was looking at these cells and they looked okay. Now they're floating all over the place."

Healthy cells normally appear flat and asymmetrical, something like a bug gone splat on a windshield. But 20 hours ago, Clayton infected these cells with an engineered virus that carries a cell death gene. Now the cells are balled up, and small pieces have broken off, a process biologists call "blebbing." As Clayton moves the plate, these balls and blebs jiggle and roll. A more powerful microscope would reveal the tightly choreographed series of steps that take place during apoptosis, which comes from the Greek word for "falling off."

It was the sight of this death choreography that first sparked Hardwick's interest in cell death, when she was a graduate student in the late 1970s and early '80s studying the measles virus. Peering into the microscope every day, she found herself wondering exactly how the measles virus killed cells. Her advisor explained that a virus assaults a cell, like a hammer smashing holes. Hardwick was not satisfied. "It didn't intuitively seem logical to me. I remember coming away thinking, something else is going on."

A small number of researchers had been studying apoptosis, but few scientists knew about or were paying attention to this research. Interest grew in the mid-'80s when scientists working in cancer cells discovered a gene that appeared to prevent cell death. They named this first anti-apoptotic gene Bcl-2. At the same time, Robert Horvitz, an MIT biologist, was elucidating the genes that controlled cell death in the nematode worm, research for which he would eventually share the Nobel Prize.

Hardwick was enthralled by such findings, and after joining the Hopkins faculty in 1986, she became one of a small band of researchers who began to explore the molecular underpinnings of apoptosis. She also began talking about cell suicide to any colleague who would listen. Programmed cell death, says Hardwick, "has been the most fascinating thing that I've worked on. It's been this mysterious [idea,] a whole new way of thinking—that cells have a purposeful destruction mechanism. And that mechanism is key to health and key to disease."

Healthy cells normally appear flat and asymmetrical, something like a bug gone splat on a windshield. A cell in the throes of apoptosis loses the protuberances of its surface.

Uncovering those mysteries has been an all-consuming passion, although she devotes some of her free time to playing violin duets with her younger son and cooking with her older son, Matt Boersma, a Hopkins neuroscience graduate student. "I'm lucky my hobby is my profession," she says. "The thrill of finding something new is so driving. It's like putting together a jigsaw puzzle." The anticipation of finding where the next piece belongs sometimes keeps her awake at night.

At first, not everyone shared Hardwick's enthusiasm for this new field. She remembers speaking to members of the National Academy of Sciences who told her, "Oh, programmed cell death, that's preposterous!"

The skeptics had good reason to question the theory, acknowledges Hardwick. "We lacked hard evidence," she says.

So, piece by piece, Hardwick and other cell death researchers went about collecting evidence, which has turned out to be an elaborate assortment of genes and proteins—the components of the cell's death machinery.

Hardwick picks up a pen and some sheets of blank paper. She starts writing, and soon she has covered the front and back side of several pages with acronyms, arrows, symbols and terms, each illustrating a piece of the complex molecular process contributed by many different researchers.

A virologist by training, Hardwick began to study cell death by investigating whether the cell death gene, Bcl-2, would alter the course of a viral infection. Working with a mosquito-borne virus called Sindbis, she demonstrated that infection triggered cell suicide in various cell types. However, Hardwick took the experiment one step further. She and her colleagues genetically engineered some cells so that they contained extra copies of the Bcl-2 gene. They then infected those cells with the Sindbis virus. This time the cells did not undergo suicide. It appeared that Bcl-2 could help cells outwit an inducement to self-annihilation.

Hardwick reported her findings in the journal Nature. Since then, she and other investigators have shown that the anti-death apparatus (a broad family of genes that includes Bcl-2) can be more active in some types of cells than in others. This might be nature's way of protecting cells that come in limited quantities. If a virus infects the epithelia of the lungs for instance, those epithelial cells can kill themselves and easily be replaced by new cells. But if a virus infects a precious neuron, that cell might activate its mechanism for repressing cell suicide.

These findings help explain why some viruses can linger in the body for months or even years. The varicella zoster virus, for example, can cause chicken pox in a child, then lie dormant for decades until it flares up again to cause the painful red rash known as shingles. Where does the virus hide all those years? In neurons, but other viruses select different parts of the body where the cell death apparatus is idling. To make matters more complicated, some viruses (such as the herpes simplex virus, which causes cold sores) are endowed with their own version of anti-death genes, which enable the virus to prevent its host from committing suicide. It's like a thief disarming a house's burglar alarm, says Hardwick.

Since these early findings on Bcl-2, Hardwick and other researchers have identified a raft of cell death genes and proteins. Ten to 20 percent of the human genome is somehow connected to programmed cell death, Hardwick says, adding, "That may even be a conservative estimate."

Ten to 20 percent of the human genome is somehow connected to programmed cell death. "That may even be a conservative estimate," says Marie Hardwick.

These discoveries are providing tools for hundreds of research endeavors in medicine and biotechnology. A sampling of projects by investigators who have collaborated with Hardwick or sought her advice:

• The cells under John Clayton's microscope come from the Asian tiger mosquito, which can transmit viruses that cause encephalitis but that do not harm the mosquito. Clayton hopes to develop a bio-friendly insecticide against these pests. The insecticide will contain a genetically engineered virus that carries a cell suicide gene that works in mosquito cells but not human cells. When the virus infects a mosquito, the gene will induce the mosquito's cells to commit suicide, thus obliterating this irritating source of disease.
   
• In the case of male infertility, reduced levels of testosterone may induce the suicide of cells that would otherwise develop into sperm, says Barry Zirkin, PhD, professor of Biochemistry and Molecular Biology and head of the Division of Reproductive Biology at the Bloomberg School. Zirkin's research on this process may lead to new treatments for infertility, as well as to new forms of male contraception.
   
• By exploiting cell death, researchers may have found a way to improve the potency of an experimental cervical cancer vaccine. The vaccine is designed to activate special immune cells called dendritic cells, explains ,T. C. Wu, professor of Pathology at the School of Medicine. But these cells normally have a short clock. By adding an anti-apoptosis gene to the vaccine, Wu has been able to extend the life of these cells and, animal studies show, improve the effectiveness of the vaccine.
   
• Likewise, Hopkins chemical engineer Michael Betenbaugh has found a way to lengthen the lifespan of the cell "factories" that the biotechnology industry uses to churn out drugs and other products. By inserting anti-death genes into these cells, Betenbaugh demonstrated he can increase the yield of these factories by 20 to 30 percent.
   
• Studies by Hardwick and School of Medicine neurologist Doug Kerr suggest that programmed cell death plays a role in a neurodegenerative disease called spinal muscular atrophy, or floppy baby syndrome. Babies with the disease have too few motor neurons, which makes them floppy and sometimes too weak to suck properly, explains Kerr, MD, PhD, who is also an assistant professor of Molecular Microbiology and Immunology at the Bloomberg School. The researchers hypothesize that the neurons are produced during development but then are destroyed when a malfunctioning protein triggers apoptosis.

If all disease involves programmed cell death, as Hardwick suggests, then one might imagine that blocking apoptosis would defy disease. Reality has proven more complicated.

"Initially, we had this exuberance about anti-apoptotic therapies," says Kerr. "It could be the miracle cure in degenerative diseases." However, it would not be that easy. Kerr once tried blocking apoptosis in cells that carry the mutation for spinal muscular atrophy. The cells lived longer than usual—but only a few days longer, and they were not healthy. "It was like preserving the undead," says Kerr. "The cells weren't really live or functioning."

Doug Kerr's hopes for a miracle cure for degenerative diseases faded when he blocked apoptosis in cells only to find "it was like preserving the undead"

Spinal muscular atrophy, says Kerr, is a complex disease that begins with a defective gene and ends with apoptosis. In between, numerous molecular events transpire, and blocking apoptosis will not fix every problem along this pathway. Kerr likens the process to a house being condemned. A deconstruction crew comes along and takes down the house piece by piece. The deconstruction, like apoptosis, gets rid of the ruined edifice. But that doesn't explain why the house—or cell—was condemned in the first place.

Kerr now says that a "multi-modal approach" will be required to treat spinal muscular atrophy: drugs that block the cell death pathway, as well as other medicines. Researchers in other diseases are coming to the same realization.

Exploiting cell death may offer more possibilities in cancer treatment, where researchers aim not to stop apoptosis but to turn on the cell death mechanism. Thousands of scientists are involved in this pursuit, says Charles Rudin, an oncologist at the School of Medicine. Rudin himself is testing tools that stimulate cell death in small-cell lung cancer.

Apoptosis is now an established subspecialty of biology, and talk of cell suicide no longer elicits raised eyebrows. Hardwick's colleagues say that she has played an important part in making that happen. "Marie Hardwick is a highly respected pioneer whose major contributions have revolved around highly innovative research on the regulation of cell death," says Doug Green, a biologist who studies apoptosis at the University of California at San Diego.

Still, Hardwick continues to push the envelope. Lately, she is exploring the notion that yeast, a single-cell organism, performs programmed cell death. "We believe that cell death is so ancient that all organisms, including bacteria, anything that is a cell, [has the tools to] perform programmed cell death," she says.

In experiments in Baker's yeast, she and Iva Ivanovska, PhD '05, currently a postdoctoral fellow at Harvard, recently demonstrated that a virus can induce cell suicide in yeast in a similar fashion to that observed in higher organisms, findings that appeared in the August 11 issue of The Journal of Cell Biology.

Small numbers of researchers are also studying apoptosis in yeast, but again, Hardwick's research broaches difficult questions. One of the main reservations is that the notion of cell suicide in yeast suggests a sort of altruism, a word that makes some biologists cringe when applied to something like yeast. How would suicide benefit a single-cell organism? You kill yourself, you're dead. End of story.

"The idea that single-cell organisms might undergo active cell death is viewed skeptically, but I think there is some acceptance of this idea," says Green. However, he says that "the jury is out" on whether the cell death in yeast qualifies as apoptosis.

Hardwick, on the other hand, says she has no doubt that what she sees in Baker's yeast is cell suicide.

No organism lives in isolation, she explains. Yeast reside among hundreds or thousands of other yeast. If one of those organisms becomes infected with a virus, the entire group is threatened. But if the infected yeast kills itself, it spares the population. "Single-cell organisms need programmed cell death not for the survival of that cell, but for the survival of the species," says Hardwick. "I think it's as old as the cell itself."