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Apoptosis Activity: Cell Death Establishes Itself As A Lively Research Field

The phenomenon of apoptosisña form of programmed cell deathñhas sprung suddenly and dramatically into scientific consciousness. While references to apoptosis now abound in scientific literature, cell biology textbooks with copyrights prior to 1992 do not contain the term in their indexes.

Before 1992, the National Institutes of Health did not list apoptosis as an area of research interest. In 1993, however, grant funding for projects whose title mentioned the word nearly doubled over the 1992 figure; funding doubled again in 1994 (not available on web site).

And yet, when Barbara Osborne, an associate professor in the molecular and cellular biology program at the University of Massachusetts, Amherst, recently traced the development of the field, she found an erratic history, characterized by little early activity followed by booming interest. She was researching the preface for a 1995 edition of Methods in Cell Biology (San Diego, Academic Press Inc.), which she coauthored with Amherst associate professor Larry Schwartz.

"Iíve traced the number of publications from 1980 to the present," reports Osborne, who searched for the keyword "apoptosis." She found that "there was nothing in the early ë80s. Then, in 1985 through 1987, a little blip [in the number of publications] appeared. Since then, itís grown logarithmically." Osborne continues to search databases for apoptosis references weekly. "When I first started in 1991, there were 300 to 400 total papers," she notes. "Now it is between 3,000 and 4,000."

Why did it take so long for apoptosis to enter the limelight? "Everybody thought about death as something you didnít want to happen, especially those of us in cell culture," says Osborne. "Sometimes it takes a while for something to sink in as being important."

The idea that life requires death seems paradoxical, but cell suicide is essential for an animal to survive. For example, without selective destruction of "non-self" T cells, an animal would lack immunity. Similarly, meaningful neural connections in the brain are whittled from a mass of cells. Apoptosis research, with roots in developmental and cell biology, genetics, and immunology, embraces this long-ignored natural law.

Programmed cell death, including apoptosis, is gene-directed. "The word comes from two Greek words, apo- and ptosis-, and the p is silent," declares Jonathan C. Busser, a researcher in the department of neurology and neurosurgery at Case Western Reserve University School of Medicine. "Apo" means "separate from" and "ptosis" means "fall from"ña description of cells that naturally, and without any inflammatory fanfare, die as part of normal development, he explains.

The Dance Of Cell Death

The steps of apoptosis are distinctive. The cell forms a tight sphere and its membrane undulates, resulting in bulges called blebs. The nuclear membrane breaks, and endonucleases clip chromosomes where the DNA peeks out from protective proteins. This occurs at 180-base intervals, so the DNA pieces are all the same size. Then the cell fragments, with enough membrane sequestering toxic cell contents to prevent inflammation at the site. Finally, nearby cells consume the remains. (In contrast to this process is necrosis, a nonprogrammed form of cell death that is a response to injury, in which the cell swells and bursts, causing inflammation.)

The pieces of the cellular death machinery are present in the cytoplasm, proven by the fact that cells whose nuclei are removed can still undergo apoptosis. A hypothesized "death signal" activates the process. Apoptosis is so fast that researchers often canít detect it, let alone sort out the sequence of events. "Once it starts, apoptosis probably takes from a few minutes to an hour," says Douglas Green, head of the division of cellular immunology at the La Jolla Institute for Allergy and Immunology in California.

On a cellular level, apoptotic cells are visualized with vital dyes and electron microscopy. On a molecular level, electrophoresis gels are used to display the tell-tale same-sized DNA pieces, which resemble ladders. Alternatively, the DNA pieces can be detected by labeling their 3¥ ends with a biotinylated thymine analog. "Cells that stain brightly are the ones with large numbers of 3¥ ends. If thereís no bright stain, thereís no apoptosis," comments Busser.

Apoptosis as part of normal development is a strategy to select certain cells for survival, sculpting a tissueís specificity. In a vertebrate embryoís limb, apoptosis carves fingers from webbing. In the developing brain it leaves behind only certain neural connections, and in the fetal thymus allows only T cells with "self" surfaces to complete development.

Later in life, apoptosis protects. Consider sunburn. A cell whose DNA is damaged by ultraviolet radiation in sunlight is either repaired or jettisoned via apoptosisñpeeling (A. Ziegler et al., Nature, 372:773-6, 1994). "Such controls ensure that any one mutated cell cannot proliferate. Without this, tumors would be incredibly common," Green explains

A Curious History

Developmental biologists have long been familiar with cell death in carving a vertebrateís digits and in insect metamorphosis. But todayís cell-death community credits a paper by University of Edinburgh researcher Andrew Wyllie and his colleagues as the seminal work in the field (J.F.R. Kerr, A.H. Wyllie, A.R. Currie, British Journal of Cancer, 26:239-57, 1972). They coined the term apoptosis, writing that it plays "a complementary but opposite role to mitosis in the regulation of animal cell populations."

The paper created little excitement initially. "It was just one of those things in the literature that stayed dormant for 10 to 15 years. Then it was gradually rediscovered and gained recognition as a generally important mechanism," reports L. Maximilian Buja, chairman of the department of pathology and laboratory medicine at the University of Texas Medical School at Houston.

What catapulted apoptosis into "hot topic" status was its meticulous demonstration in a tiny worm, followed by identification of death genes in other organisms (J. Sulston, H.R. Horvitz, Developmental Biology, 56:110-56, 1977). In the 1980s, the term "programmed cell death" was almost synonymous with Caenorhabditis elegans, the tiny, transparent nematode worm whose cell-death program removes precisely 131 of 1,090 cells to form the adult.

"What has pushed the field forward is Bob Horvitzís work, which allowed us to look at the process in the worm," says Osborne, who spent a sabbatical year in 1992 in the lab of Horvitz, a Howard Hughes Medical Institute (HHMI) investigator and a professor of biology at the Massachusetts Institute of Technology. "The fact that you have a certain number of cells and can trace their developmental fates and see what happens, and watch under the microscope and predict which cells will die, then isolate genes, has made the field blossom and flourish."

Meanwhile, little was known about cell death in other types of animals. When David Hockenbery, Stanley Korsmeyer, and their HHMI group at the Washington University School of Medicine in St. Louis discovered that the proto-oncogene bcl-2 blocks programmed cell death (D. Hockenbery et al., Nature, 348:334-6, 1990), this and other work on bcl-2 refocused attention on apoptosis, contributing to the second blip of interest in the early l990s.

Soon, researchers using worm genes with mutations called "ced" (for cell death abnormal) as probes identified death genes in other animals. "It was a great advance to realize that some [apoptosis] genes in the nematode are similar to genes in mammals," says Hermann Steller, an associate professor of neurobiology and an HHMI investigator at MIT who recently discovered an apoptosis gene in Drosophila melanogaster (K. White et al., Science, 264:677-82, 1994).

Pieces Of Pathways

Little is known about the many-tiered genetic control of apoptosis. Most apoptosis genes under investigation turn the process on or off.

"Apoptosis on" genes include ced-3 and ced-4 in C. elegans, and ICE and p53 in mammals. Expression of ced-3 and ced-4 is necessary for the cell death of normal worm development. The gene ced-4 encodes a novel protein, but ced-3 is a homolog of ICE (interleukin-1b converting enzyme).

Experiments demonstrate the link between the gene ICE and apoptosis. Rat fibroblasts genetically engineered to overproduce ICE die by apoptosis, and phagocytes gain ICE after gobbling apoptotic cells. Recently, Junying Yuanñan associate professor in the department of medicine at Harvard Medical Schoolñand her colleagues, working with a chicken neuron cell culture, found the inhibiting ICE activity prevents the cells from dying when their supply of nerve growth factor is blocked (V. Gagliardini et al., Science, 263:826-8, 1994).

Another "apoptosis oníí gene receiving much attention is p53. The gene, which encodes a transcription factor and is common in many human cancers, mediates cellular responses to some environmental damage. The p53 protein either temporarily halts cell division so the cell can repair altered DNA, or sends the cell to an apoptotic death.

"How p53 makes that choice is the $64,000 question. Maybe there is a threshold. If damage is minor, the cell takes the time to repair it. But if damage is above threshold, it bails out, choosing apoptosis. The threshold may be different in different cell types," says Alexander Kamb, director of research at Myriad Genetics Inc. in Salt Lake City, Utah. Adds Steven Schreiber, an assistant professor of neurology at the University of Southern California School of Medicine: "Maybe the choice of cell death or arrest of the cell cycle depends on the proliferative capacity of the cellñif it has a certain number of cycles to go. Or, it may depend upon the stage of the cell cycle when the damage occurs."

Work on p53 reveals that there are different means to an apoptotic end. "Our work with Tyler Jacks [a professor of biology] at MIT shows that p53 only kills thymocytes when the inducer to death is radiation," says Osborne. Other thymic inducers include glucocorticoids and cross-linking T-cell receptors. In nerve tissue, two cell-surface proteins, TNF R1 and APO-1/Fas, induce apoptosis when they bind their ligands.

The bcl-2 gene is an "apoptosis off" control. In 1992, Korsmeyer discovered that bcl-2 is the mammalian equivalent of ced-9, a worm anti-death gene (S.J. Korsmeyer, Blood, 80:879-86). The proto-oncogene is an apoptosis "brake" in the skin. The gene is expressed in the basal layers, where stem cells must divide to supply more cells. In the upper layers, lack of bcl-2 protein permits apoptosis, preventing tumor formation. The bcl-2 protein binds a protein called bax. The bcl-2/bax ratio is critical to a cellís fate. If bcl-2 is in excess, all available bax is bound, apoptosis is blocked, and the cell lives. If bax is in excess, all bcl-2 is bound, the brake is released, and the cell dies (Z. Oltvai, C. Milliman, S. Korsmeyer, Cell, 74:609-14, 1993).

Certain viruses also have apoptosis brake genes. "This keeps the cell it infects from committing suicide," says Steller. Such genes are found in adenovirus, Epstein-Barr virus, African swine flu virus, and vaccinia.

Clinical Applications

Apoptosisí ties to the number of cells in certain organs and tissues suggest applications in correcting medical problems stemming from particular cellular excess or deficiency. "Weíve always thought of cancer as a proliferative process. Now thereís a whole new way of thinkingñthe absence of cell death sets the stage for proliferation," says Buja.

Kamb foresees applying knowledge about apoptosis to monitoring cancer treatment: "If you give chemotherapy that works through the apoptotic pathway, and can show that the tumor is not apoptosis-competent, youíd know that youíre just poisoning the patient. Paying attention to apoptosis competency in tumors, diagnostically and prognostically, may provide a way to tailor therapy."

By Ricki Lewis
Ricki Lewis is a textbook author and science writer based in Scotia, N.Y.

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