The fact that apoptosis has been the subject of increasing attention and research efforts was highlighted by the award of the 2002 Nobel Prize in Physiology or Medicine to Sydney Brenner (Great Britain), H. Robert Horvitz (US) and John E. Sulston (GB) "for their discoveries concerning genetic regulation of organ development and programmed cell death" (see [1] ).
A fascinating apoptotic process, resulting from stress or toxicity that damages the genome in the cell nucleus, is cell suicide triggered by the nuclear enzyme poli(ADP-ribose) polymerase-1, or PARP-1. This enzyme plays a crucial role in maintaining genomic integrity; massive activation of PARP-1 can deplete the cell of energy-providing molecules, an event that sends signals from the nucleus for the mitochondrion to start the apoptotic process (see the Perspective "PARP-1 -a Perpetrator of Apoptotic Cell Death?", by Alberto Chiarugi and Michael A. Moskowitz, in Science, Vol. 297, No. 5579, p. 200, and the research report by Seong-Woon Yu, et al., in p. 259, in the same issue).
Research on chick embryos -- specifically on chick neural tube development -- has suggested how selective cell proliferation, combined with selective apoptosys, sculpts developing tissues in vertebrates. During vertebrate embryo development, structures called the notocord and the floor plate secrete a gradient of the signaling molecule Sonic hedgehog (Shh), and it is this gradient that directs cells to form patterns in the embryonic neural tube: cells that receive Shh in a receptor in their membranes called Patched1 (Ptc1) survive and proliferate; but, in the absence of Shh, one of the ends of this same Ptc1 receptor (the carboxyl-terminal, inside the membrane) is cleaved by caspase-3, an action that exposes an aptotosys-producing domain. (See the Perspective "Longing for Ligand: Hedgehog, Patched, and Cell Death", by Isabel Guerrero and Ariel Ruiz i Altaba, in Science Vol. 301, No. 5634, p. 774; and the research report "Inhibition of Neuroepithelial Patched-Induced Apoptosis by Sonic Hedgehog" by Chantal Thibert, et al., in p. 843 of that same issue, Aug. 8, 2003).
Research like the one carried out by Thibert and her colleagues has begun to clarify some of the fundamental aspects of ontogeny, or the development of organisms from fertilized eggs to fully-developed animals and plants. It has also suggested specific answers to why normal cells carry out apopotosis when they end up outside the places they should be in body tissues.
Homeostasis is achieved when the rate of mitosis (cell proliferation) in the tissue is balanced by cell death. If this equilibrium is disturbed, either of two things happen:
In the studies on mouse embryos lacking PS receptors ("PSR knockout mice")
conducted by Li and colleagues, un-ingested cells undergoing apoptosis accumulated in the brain and lungs, leading to neonatal lethality. These studies show how critical is the role of PS receptor (PSR) in the development of complex organisms such as mammals.
Apoptotic messages from outside the cell (extrinsic factors) will be briefly desribed in the next section of this article. (For a detailed description of an extrinsic apoptotic pathway see "The Fas Signaling Pathway: More Than a Paradigm", by Harald Wajant, in Science, Vol. 296, No. 5573, p. 1635, May 31, 2002).
Apoptotic messages from inside the cell (intrinsic factors) emerge from mitochondria.
Tumor necrosis factor (TNF), a 157 amino acid inter-cellular signaling molecule (cytokine), is the major extrinsic mediator of apoptosis. The cell membrane has two specialized receptors for TNF: TNF-R1 and TNF-R2. The binding of TNF to TNF-R1 has been shown to fire-off the pathway that leads to activating the caspases (see "TNF-R1 Signaling: A Beautiful Pathway", by Guoqing Chen and David V. Goeddel, in Science, Vol. 296, No. 5573, p. 1634).
Downstream from TNF activation --at least in mammalian cells-- the proapoptotic molecules BAK and BAX are required in order to make the mitchondrial membrane permeable for the release of caspase activators. Just how BAX and BAK are controleld under the normal conditions of cells that are not undergoing apoptosis, is incompletely understood. But it has been found that a mitochondrial outer-membrane protein, VDAC2, interacts with BAK to keep this potentially lethal apoptotic effector under control. When the death signal is received, products of the activation cascade --such as tBID, BIM or BAD-- displace VDAC2: BAK and BAX are activated, and the mitochondrial outer-membrane becomes permeable. This results in the release of caspase activators, including cytochrome c (see "VDAC2 Inhibits BAK Activation and Mitochondrial Apoptosis", by Emily H.-Y. Cheng, Tatiana V. Sheiko, et al., in Science, Vol. 301, No. 5632, July 25, 2003, p. 513).
The whole process requires energy and a cell machinery not too damaged. If the cell damage is between certain levels, the cell can start the earliest events of apoptosis and then continue with a necrosis.
The link between TNF and apoptosis shows why an abnormal production of TNF plays a fundamental role in several human diseases, especially (but not only) in autoimmune diseases, such as diabetes and multiple sclerosis.
Work carried our by Takaoka and colleagues has clarified the role played by interferon in the treatment of some forms of human cancer, and has increased knowledge on the link between p53 and IFN alfa/beta. The p53 response not only contributes to tumor suppression, but is also essential in evoking an apoptotic response to viral infection and consequent damage to the cell's reproductive cicle.
The apoptotic pathway was found to be dramatically restored in H460 cells with a Smac peptide (SmacN7) that targets IAPs. Yang and her team successfully developed a SmacN7 peptide that selectively reversed apoptosis resistance --and, hence, tumor growth-- in H460 cells in mice.
Biologists had long suspected that mitochondria originated from bacteria that had been incorporated as endosymbionts (that is, a living body "living together inside") of larger, eukaryotic cells. It was Lynn Margulis who, since 1967, began championing this theory, that has since been widely accepted (see "The Birth of Complex Cells", by Christian de Duve, Scientific American Vol. 274, 4, April, 1996). The most convincing evidence for this theory is the fact that mitochondria have their own DNA, and are equipped with their own genes and replication apparatus.
This evolutionary step must have been more than risky for the eukaryotes that began to engulf energy-producing prokaryotic bacteria, the ancestors of mitochondria. The drama is still enacted today in our own white blood cells (which, it must be said, are much better equipped to entrap and destroy bacteria that intend to invade our bodies). Most of the time, invading bacteria are destroyed by the white blood cells; but, oftentimes, the chemical warfare waged by the prokaryotes succeeds, with the known consequences of infection, and the resulting damage.
One of those rare events in evolution, about two billion years before the present, must have made it possible for certain eukaryotes and energy-producing prokaryotes not only to coexist, but to mutually benefit from their symbiosis.
In a very real and immediate sense, it can be said that eukaryotic cells live poised between life and death, because mitochondria still retain their repertoire of molecules that can trigger cell suicide (see the afore-quoted article by Chiarugi and Moskowitz, in Science 297, p. 200). Given certain signals or insults to cells—such as feed-back from neighbors, stress or DNA damage—mitochondria release caspase activators that produce the cell-death-inducing biochemical cascade.
As has been previously explained at the beginning of this article, however, this fine equilibrium between life and death that all of us eukaryotic beings carry most intimately and deeply, is essential to life.
Functions of apoptosis
Cell damage or infection
Apoptosis can occur, for instance, when a cell is damaged beyond repair, or infected with a virus. The "decision" for apoptosis can come from the cell itself, from its surrounding tissue or from a call that is part of the immune system. If a cell's capability of apoptosis is damaged (for example, by mutation), or if the initiation of apoptosis is blocked (by a virus), a damaged cell can continue dividing without restrictions, developing into cancer.Immune cell regulation
Some cells of the immune systems, the B cells and T cells, can become autoreactive, attacking healthy body cells. These are destroyed via apoptosis. New T cells are tested for autoimmune reactions within the thymus so that they do not attack healthy body cells right away. About 95% of the freshly produced T cells are killed right away via apoptosis due to autoimmune reactions.Development
Programmed cell death is an integral part of metazoa (multicellular animals) tissue development, and it does not elicit the inflammatory response which is characteristic of necrosis (see "Mechanisms and Genes of Cellular Suicide", by Hermann Steller, Science Vol. 267, Mar. 10, 1995, p. 1445). In other words, apoptosis does not resemble the sort of reaction that comes as a result of tissue damage due to accident or pathogenic infection. Instead of swelling and bursting --and, hence, spilling their internal contents into extracellular space--, apoptotic cells and their nuclei shrink, and often fragment. In this way, they can be efficiently phagocytosed (and, as a consequence of this, their components reused) by macrophages or by neighboring cells.Homeostasis
In the adult organism, the number of cells within an organ or tissue has to be constant within a certain range. Blood and skin cells, for instance, are constantly renewed by their respective progenitor cells; but this proliferation has to be compensated by cell death. This is called homeostasis, although some authors and researchers like Steven Rose and Antonio Damasio have suggested homeodynamics as a more accurate and elocuent term (see Damasio: The Feeling of What Happens, Harcourt Brace & Co., New York, 1999, p. 141).
Both states can be fatal or highly damaging. For instance, misregulation of Hedgehog (Hgg) protein signalling (see previous section, Development) has been implicated in several forms of cancer. Hgg, which conveys an anti-apoptotic signal, has been found to be overexpressed in pancreatic adenocarcinoma tissues (see "Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis" by Sarah P. Thayer et al., Nature Vol. 425, pgs. 851-856, Oct. 23, 2003).Apoptotic process
Morphology
A cell undergoing apoptosis shows a characteristic morphology that can be seen under a microscope:
(See "Apoptosis in the Pathogenesis and Treatment of Desease", by Craig B. Thompson, in Science, Vol. 267, p. 1456, Mar. 10, 1995.)Biochemical signals for safe disposal
The dying cells that have just been described display "eat me" signals, like phosphatidylserine (PS, a phospholipid from the inner cell-membrane). Phagocytic scavengers, such as macrophages, have specialized receptors that recognize PS and carry out their disposal job in an orderly manner without eliciting an inflammatory response. (See the Perspective "Eat me or die", by Savill et al., in Science, Vol. 302, p. 1516, Nov. 28, 2003, and the corresponding research articles on new work by Li et al., and Wang et al. in the same issue of Science.)Biochemical reactions
Most apoptosis-inducing messages, from both outside and inside the cell, target a central death signal. This signal activates ICE-proteases, which initiate and perform part of the apoptosis program.Biochemical execution
Apoptosis is executed by enzymes called caspases (see "Controlling the Caspases", by Stephen W. Fesik and Yigong Shi, in Science, Vol. 294, No. 5546, p. 1477, November 16, 2001), which are normally suppressed by IAP (inhibitor of apoptosis) proteins. When a cell receives an apoptotic stimulus, IAP activity is relieved after SMAC (Second Mitochondria-derived Activator of Caspases, also called DIABLO), a mitochondrial protein, is released into the cytosol.Apoptosis and the role of interferon in tumor suppression
Akinori Takaoka et al. reported in the July 31, 2003 (Vol. 424, number 6948) issue of Nature on the "Integration of interferon-alfa/beta signalling to p53 responses in tumor suppression and antiviral defence". Their research shows that transcription of the p53 gene is induced by interferon alfa and beta (IFN-alfa/beta), resulting in the increase of p53 protein level. The latter is a powerfull tumor suppressor, and is considered as a negative-growth and anti-oncogenic factor. Increasing evidence links cancer with defective apoptotic pathways
Liling Yang et al. reported in the Feb. 15, 2003, issue of Cancer Research the results of their work in the role played by a defective death signal in a type of lung cancer cells called NCI-H460 (human non-small cell lung cancer cells). They found that cross-linked inhibitor of apoptosis (XIAP) proteins are overexpressed in H460 cells. XIAPs bind to the processed form of caspase-9, and supress the activity of apoptotic activator cytochrome c (the apoptosome produced by the mitochondrion). Role of apoptotic products in tumor immunity
An interesting case of re-use and feed-back of apoptotic products was presented by Matthew L. Albert in a research article that won him an Amersham Biosciences & Science Prize for Young Scientists in Molecular Biology, and published in Science Online in December, 2001. Albert described how dendritic Cells, a type of antigen-presenting cells, phagocytose (that is, engulf) apoptotic tumor cells. Upon maturation, these dendritic cells present antigen (derived from the apoptotic corpses) to killer T cells, which are then primed for the eradication of cells undergoing malignant transformation. This apoptosis-dependent pathway for T cell activation is not present during necrosis, and has opened exciting posibilities in tumor immunity research. Evolutionary origin of the apoptotic process