© Florence Folmer, November 2000

Apoptosis - a review

Key words: Apoptosis, C. elegans, D. melanogaster, caspases, bcl-2 family members, cancer therapy

Apoptosis, a process of programmed and regulated cell destruction which is frequently used by multicellular organisms to discard cells that are in excess, is currently one of the hottest topics of modern biology. Interest and research activity in the area of apoptosis has expanded exponentially during the last two decades1,2.

Developmental biologists have long been familiar with the existence of cell death mechanisms involved in the carving of the vertebrate's digits and during insect metamorphosis1,3. The term "apoptosis" comes from the Greek words "apo", which means "separate from" and "ptosis", which means "fall from", and it describes the process by which cells naturally die as part of their normal development. The term was used for the first time in 1972 by Kerr, Wyllie, & Currie4 as a mean of distinguishing a morphologically distinctive form of cell death which was associated with normal physiology1,3,4,5. Apoptosis was distinguished from necrosis, which was associated with acute injury to cells1. The paper published by Kerr et al. in 1972 created little excitement, and the study of programmed cell death remained dormant until the meticulous demonstration of the process of apoptosis in the nematode Caenorhabditis elegans and the discovery of apoptosis-genes by Bob Horvitz in the early 1980's. In the early 1990's, apoptosis gained further attention when David Hockenbery et al. discovered that the proto-oncogene bcl-2 blocks programmed cell death. Soon, researchers using C. elegans genes with mutations called "ced" (for cell death defective) as probes started to identify apoptosis genes in many different organisms, and currently an enormous amount of research effort is put into the understanding of the mechanism of programmed cell death, and into potential applications of apoptosis-regulation in medicine and in developmental biology1,3,6,7. Since 1993, the U.S. National Institutes of Health have been considering apoptosis as a promising area of research, and the grant funding for apoptosis related research has been constantly increasing3.

Apoptosis is a complex phenomenon of related morphological and biochemical processes that can vary with tissue and cell type3. Morphologically, apoptosis is characterised by a series of structural changes in dying cells, such as blebbing of the plasma membrane, condensation of the cytoplasm and nucleus, dilatation of the endoplasmic reticulum, and cellular fragmentation into membrane apoptotic bodies3. Biochemically, apoptosis is characterised by the condensation and the degradation of chromatin, by the cleavage of DNA into nucleosomal size fragments, and by rapid engulfment of the dying cell by macrophages3,6,7.

In the present paper, I review the methods used to study apoptosis, the mechanisms involved in apoptosis, the role of apoptosis in cellular and DNA damage, the interactions between apoptosis and the cell cycle, the role of apoptosis in development, and, finally, the future prospects of the study of apoptosis.

Caenorhabditis elegans, Drosophila melanogaster, and the models used to study apoptosis

The nematode C. elegans is the model organism used in the early 1970's by Horvitz et al. to define the genetic blueprint of the cell death pathway1,3,4,5. C. elegans shares with D. melanogaster, which became the second model organism for the genetic analysis of apoptosis, the accessibility to rigorous genetic analyses. Hence, both C. elegans and D. melanogaster offer unique opportunities to study the mechanisms by which cells undergo apoptosis, and to investigate how the cell death program is regulated by many different signalling pathways6,7.

During normal development of C. elegans, 131 of the 1090 cells die in every worm. Horvitz et al. have developed a model allowing them to trace the developmental fates of the different cells of the worm, to become able to predict which cells will die by watching them under the microscope, and to isolate the genes responsible for the cell death. In this way, Horvitz et al. have discovered that mutations in any of the three genes ced-3, ced-4 and ced-9 prevent the 131 naturally occuring cell deaths3. Since there are many similarities between the two apoptosis-promoting genes ced-3 and ced-4 of C. elegans and ICE and p53, respectively, in mammals, as well as between the apoptosis-inhibiting gene ced-9 of C. elegans and bcl-2 in mammals, Caenorhabditis elegans is a good model for the study of apoptosis3,6,8.

The dying cells can be easily visualised under the microscope by staining unfixed tissues or live embryos with the vital dye Acridine orange6,8. Dual laser cytometry (at =488 nm, using 7-AAD; U.V. staining, using Hoechst 33342 fluorescent dye), as described by Schmid et al., is a rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis. Apoptotic cells bind can be identified since they bind much more Hoechst 33342 fluorescent dye and consequently are about 5-7 times brighter than normal cells. Necrotic cells also bind to Hoechst 33342, but in contrast with apoptotic cells, which lack of staining with 7-amino-actinomycin-D, necrotic cells take up the red-emitting dye 7-AAD very easily9. Further more, apoptotic cells have a slightly smaller forward light scatter than normal cells9. TdT-mediated dUTP nickend labelling (TUNEL) is an additional method used for the identification of apoptotic cells6.

In D. melanogaster, large numbers of cells die both during embryonic development and during metamorphosis. In order to identify genes involved in apoptosis, White et al. have examined embryos, homozygous for previously identified chromosomal deletions, for defects in cell death. Screening a collection of 140 deletion strains that spanned approximately half of the Drosophila genome has allowed White et al. to find out that one region, termed cytosolic position 75C, is essential for virtually all cell deaths that normally occur during embryogenesis. Molecular analysis of the 75C region has revealed three cell death genes in the H99 interval, namely reaper, hid, and grim, which induce apoptosis via a caspase pathway when ectopically expressed. When expressed under the control of the eye-specific pGMR promoter, reaper, hid, or grim cause cell death, resulting in eye ablation. These eye phenotypes are highly sensitive to the transgene dosage, so that, at intermediate levels of expression, reduced and roughened eyes are generated. Under conditions of partial eye ablation, cells are highly sensitive to alternations in the dosage of cell death genes acting downstream of reaper and hid. This permits very simple and efficient F1 screens for genetic modifiers of reaper- and hid- mediated cell killing: mutations that promote apoptosis can be identified as enhancers of eye defects, while mutations that inhibit death supress this phenotype. Because the Drosophila eye is a non-essential and easily score-able tissue, it is possible to screen large numbers of mutagenised flies and isolate not only inactivating alleles, but also rare gain-of-function mutations in cell death genes. In addition, new techniques in genetics and in developmental biology of Drosophila are constantly elaborated, leading to new experimental models which will help advance the analysis of the role of cell death genes in development6.

C. elegans and D. melanogaster are, with no doubt, the two model organisms, or research tools, that allowed the exponential increase in the study and the understanding of apoptosis3,4,6. Nevertheless, new experimental models are under development, and might be very promising as well. In 1999, Matsuyama et al. suggested to use yeast as model organisms to study apoptosis. Although yeast does not possess the proteases ultimately responsible for the morphological events recognised as apoptosis, the simple unicellular eukaryotes can serve as a powerful tool for apoptosis researchers. Indeed, ectopic expression of several human and animal apoptosis proteins in either budding or fission yeast results in phenotypes that create opportunities for genetic screening10. As explained by Schmid9, human thymocytes constantly undergo cell death, and the rate of apoptosis increases after exposure to glucocorticoid dextramethasone. Thymocytes can hence be considered as potential models for the study of apoptosis.

Recently, considerable amount of research has focused on identifying the molecular sensors or triggers of apoptosis. The best studied mammalian proteins that fit the classification of sensors or triggers of apoptosis are the tumour suppressor p53 and the Fas ligand/receptor system. Studies using p53 minus cell lines transfected with a temperature sensitive mutant of p53 have clearly shown that wild-type p53 function lowers the threshold for inducing apoptosis following genotoxic damage. Studies on the Fas ligand/receptor system have been done, i.e. using antibodies against Fas. In 1994, Cifone et al. discovered that the production of ceramide via activation of an acid sphingomyelinase may play an important role in the Fas signalling system, and that the addition of synthetic C2-ceramide alone to culture medium can mimic Fas activation and trigger apoptosis6.

Recently, Samejima has compared cytoplasmic extracts from chicken cells at various stages along the apoptotic pathway in order to try to understand the mechanisms of apoptosis11. At the beginning of this year, Matsuyama et al. suggested to study changes in intramitochondrial and cytosolic pH in order to detect early events that modulate caspase activation during apoptosis12.

Wride has also presented the developing lens of vertebrate eyes as a useful model system for researchers interested in apoptosis. Programmed removal of nuclei and other organelles from the lens fibre cells ensures that an optically clear structure is created, and the morphology of the degenerating nuclei is similar to that observed during apoptosis13.

The different experimental models mentioned above are summarised in table 1. Many of these models are recent, and it is still to early to draw conclusions regarding the results which have been obtained, and to understand the process of programmed cell death thoroughly. Nevertheless, the efforts put into apoptosis-related research during the last decade have allowed to understand the basic mechanisms of apoptosis, which are presented in the next section.

Table 1: The major experimental models currently used to study apoptosis

  • The nematode C. elegans (catalyst of apoptosis research; similarities of ced-3 and of ced-4 with human ICE and p53, respectively)
  • The fruitfly D. melanogaster
  • Observation and quantification of cell death by microscopy, by dual laser cytometry, and by TUNEL labelling
  • F1 screening for genetic modifiers of reaper- and hid- mediated cell killers in developing embryos
  • Yeast cells; thymocytes
  • The study of molecular sensors or triggers of apoptosis, i.e. p53 and Fas-receptors/ligands, using antibody labelling
  • The study of cytoplasmic extracts
  • The developing lens of vertebrate eyes

The mechanisms involved in apoptosis

Apoptosis is a genetically mediated process which can be divided into three phases: an induction phase, which is still poorly understood, and which depends on the specific intracellular or extra-cellular death-inducing signals, an effector phase, during which the "central executioner" is activated and the cell becomes committed to die, and a degradation phase, during which the cells acquire the biochemical and morphological features of end-stage apoptosis14. The two main families of "central executors", or apoptotic regulators taken into consideration are the caspases , or ICE-like proteases (cell death promoters), represented by caspases 1-10 in mammals, by ced-3 in C. elegans, and by dcp-1 and drICE in D. melanogaster; and the Bcl-2 family members, represented by Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 (cell death inhibitors), by Bax, Bcl-xs, Bad, Bak (cell death promoters) in mammals, and by ced-9 (cell death inhibitor) in C. elegans5,6,15. A third family, which is composed of ced-4 homologues, is thought to facilitate the interactions between caspases and bcl-2 members16. In mammals, death-domain proteins such as CD95 (Apo-1/Fas), TNFR1, FADD/MORTI, TRADD, and RIP are also known to play an important role in the regulation of apoptosis, and in many cell types, and caspase-independent apoptosis is quite frequent2,6,13,14.

Caspases use a cysteine protease mechanism to selectively cleave a restricted set of target proteins at aspartic acid residues2,8,13. As many other proteases, caspases are synthesised as zymogens which contain an N-terminal prodomain and p20 and p10 domains which are found in the mature enzyme. Most caspases are activated by proteolytic cleavage, by active caspases, of the zymogen between the p20 and p10 domain, and usually also between the prodomain and the p20 domain. This caspase cascade is a useful method to amplify and to integrate pro-apoptotic signals. In addition, two other mechanisms are known to activate caspases. Caspases can be activated by forced crowding of zymogens, as it has been shown with Caspase-8: Upon ligand binding, death receptors such as CD95 aggregate and form membrane-bound signalling complexes which then recruit, through adapter proteins, several molecules of procaspase-8, resulting in a high local concentration of zymogen. Under these crowded conditions, the low intrinsic protease activity of procaspase-8 is sufficient to allow the various proenzyme molecules to mutually cleave and activate each other. The activation of other caspases, such as caspase-9, requires association of the caspase with a regulatory protein (i.e. a protein cofactor). Caspase-9 activation, for example, requires the formation of an Apaf-1/caspase-9 complex. Apaf-1 is considered as an essential regulatory subunit of a caspase-9 holoenzyme, which is often referred to as the apoptosome. Most caspases contain a protein-protein interaction module in their prodomain, which allows them to bind to and associate with their upstream regulators. Caspase-8 and -10 contain a death-effector domain (DED), and caspase-2 and -9 contain a caspase activation and recruitment domain (CARD). In some cases, cells can be rescued from caspase-induced death by potent caspase inhibitors known as the inhibitors-of-apoptosis proteins (IAP)2. Once activated, the caspases are capable to lyse their target proteins, and thereby to destroy their target cells or organelles, which can then be degraded by phagocytes2,8.

The Bcl-2 family proteins are highly related with each other in one or more specific regions commonly referred to as Bcl-2 homology (BH) domains. One way by which Bcl-2 family members, the BH1 and BH2 domain proteins, act as apoptosis regulators is by forming heterodimers of apoptotic promoters/inhibitors, giving the cells an overall sensitivity or resistance, respectively, to death2. But the main function of the Bcl-2 members is to form channels that facilitate protein transport through the outer mitochondrial membrane 15. Indeed, in addition to their function as the cell's powerhouse, mitochondria sequester pro-apoptotic proteins such as cytochrome c (which is required for the activation of caspase-9 in the cytosol), AIF (a flavoprotein with potent apoptotic activity), Smac/DIABLO, and several procaspases, including procaspase-2, -3, and -9. Bcl-2 family members can insert themselves into the lipid bilayer of the mitochondrial outer membrane, oligmerise, and form channels with discrete conductance, which potentially allow the pro-apoptic proteins mentioned above to penetrate into the cytosol2. The Bcl2-members can also recruit other mitochondrial outer membrane proteins into forming a large pore channel such as the voltage-dependent anion channel (VDAC)2. A third possibility is that Bcl-2 members alter the mitochondrial physiology such that the organelle swells, resulting in the physical rupture of the outer membrane and the release of intermembrane proteins into the cytosol2. Finally, Bcl-2 proteins could also influence mitochondrial homeostasis indirectly through modulation of other mitochondrial proteins such as the VDAC protein, which is a subunit of the mitochondrial permeability transition pore (PTP), a large channel whose opening results in a rapid loss of membrane potential and organellar swelling. Opening of the PTP complex quickly leads to the release of the pro-apoptotic proteins, and consequently to apoptosis2.

In order to insure that cell will either fully commit to death or completely abstain from it, the apoptotic pathway contains a number of amplification steps and positive feedback loops. For example, the caspase cascade leads to a rapid amplification of apoptotic activity, and there is likely to be positive feedback between caspase activation and cytochrome c exit from mitochondria. In order to minimise the risk of undesired full apoptotic death of the cells resulting from accidental perturbation, the positive feedback loops are buffered by IAPs2.

The degradation phase of apoptosis consist in the reduction of the cells to shrivelled corpses which are rapidely devoured by neighbouring phagocytes15. Nuclear shrinking and budding is thought to result from caspase-mediated cleavage of the nuclear lamins, and the loss of overall cell shape is probably caused by the cleavage of cytoskeletal proteins such as fodrin and gelsolin (substrates for caspase-3 family members such as CPP32, apopain, and YAMA)2,11,17.

A schematic overview of the main mechanisms involved in apoptosis is represented in figure 1 and in table 2. In reality, the process of apoptosis is most certainly a lot more complex than as presented above, and the majority of the apoptotic subprogrammes are still poorly understood2.

Table 2. Possible mechanisms of action of Bcl-2 family members

  • Heterodimerisation between pro- and anti-apoptotic family members
  • Direct regulation of caspases via adapter molecules
  • Formation (optionally through interaction with other mitochondrial proteins)

of pores in the mitochondrial membranes through which cytochrome c,...

can escape into the cytosol

  • Oligomerisation leading to weakly selective ion channels

(after: Hengartner, M.O., 20002)

Cell death as a response to cellular or DNA damage

Procaspases and other proteins required to carry out apoptosis are made continuously by healthy cells. If a cell, or even an organelle, is stressed or damaged, procaspases can

Figure 1. The apoptotic pathways existing in mammalian cells

(digital version still under construction)

The death-receptor pathway:

Binding of CD95L to CD95 induces receptor clustering and the formation of a death-inducing complex which recruits, via FADD, multiple pro-caspase 8 molecules. This results in caspase-8 activation, which initiates the caspase-activation cascade.

The mitochondrial pathway:

Bcl-2 family members are activated by DNA damage,... Pro- and anti-apoptotic Bcl-2 family members meet at the surface of mitochondria, where they compete to regulate cytochrome c exit. If pro-apoptotic members win, AIF, Cyt. c, and Smac/DIABLO are released from the mitochondrion. Cyt. c associates with Apaf-1 and Pro-caspase 9 to form the active apoptosome, which stimulates the caspase-activation cascade.

The activation of the PTP-complex or of the Bcl-2/Bax complex also cause leakage of Cyt. c, AIF, and Smac/DIABLO into the cytoplasm. In addition, the opening of the PTP-complex has major effects on the energy metabolism, and it causes generation of reactive oxygen (RO) species (i.e. superoxide) - which are highly toxic. The increase of [RO] stimulates Pro-apoptotic mechanisms such as the opening of the PTP-complex (positive feedback). Cyt. c release from the mitochondria interrupts the transfer of electrons between respiratory chain complexes III and IV, thereby comprising respiratory function and causing the generation of superoxide ions.

Convergence of the death-receptor pathway and the mitochondrial pathway:

The two pathways converge at the level of caspase-3 activation, which is inhibited by IAP. The mechanisms downstream of caspase-3 result in the ordered dismantling and removal of the cell.

____________________________________________________________________

after: Hengartner, M.O.2, Green, D. & Kroemer, G.14, and Yuan, J. & Yanker, B.A.19

become activated with or without a stimulating signal. This phenomenon has been shown to take place in animals, plants, and bacteria8. When mitochondria are damaged, i.e. by a toxic drug, they can release cytochrome c into the cytoplasm, where it initiates a caspase-9 cascade leading to apoptosis. Damaged DNA or misfolded proteins in the endoplasmic reticulum can induce apoptosis via the cancer-suppressing protein p531,8. In the event of viral infection, lymphocytes become activated and induce the infected cells to kill themselves. Lymphocytes proceed by the secretion of perforins onto the surface of the infected cells, as well as by the production of a protein (Fas ligand) that binds to receptors (Fas) on the surface of the target cells, causing the aggregation of receptors into a cluster at the cell border, followed by the recruitment and the activation of procaspase-8 molecules leading to cell death. This CD95-CD95L killing mechanism is used, for example, to eliminate normal activated lymphocytes after they have done their job, which helps to terminate immune responses8,18. During ischaemic brain injury, mitochondria play an important role in the transmission of apoptotic signals and in the activation of apoptosis-resistant caspase-3 and -11. It has been shown that, in the case of ischaemic brain injury, only neurons that maintain a minimum level of metabolic activity can undergo apoptosis19.

The interactions between apoptosis and the cell cycle

Tissue homeostasis requires a balance between cell proliferation and death. Apoptosis and proliferation are linked by cell cycle regulators such as c-myc, cyclin D3, p53, pRb, and E2F. Decreased expression of c-myc and cyclin D3, for instance, induce glucocorticoid-dependent G1 growth arrest and apoptosis in transformed lymphoid cells5,20.

The role of apoptosis in development

It is now clear that physiological cell death is an essential component of animal and plant development, important for the establishment and the maintenance of tissue architecture. Amongst the best illustrations of the role of apoptosis in development are the elimination and the remodelling of transitory organs and tissues such as the carving of the vertebrate's digits during embryogenesis, the disappearance of the tadpole's tail during metamorphosis, the loss of neurons in the developing cerebral cortex, and the loss of 10% of its somatic cells by C. elegans during ontogeny11,20,21. Apoptosis also plays an important role in the development of mature T and B lymphocytes via the CD95-CD95L system. It insures that any autoimmune lymphocytes are discarded from the immune system18.

A variety of transcriptional mechanisms are involved in the regulation of apoptotic processes involved in the development. The processes involved in developmental apoptosis often rely on extra-cellular interactions. The disappearance of the tadpole's tail, for instance, is triggered by a surge of thyroid hormone in the bloodstream. Steroid hormone receptors are critical controllers of apoptosis in many mammalian tissues including the mammary gland, the prostate, the ovary and the testis. Interdigital cell death is determined by the transcriptional readout of the transforming growth factor- signalling pathway8,22.

Apoptosis is an essential component of most developmental abnormalities and human diseases and, in many cases, it is the underlying cause of the resulting pathology22. Hence, it is very important to continue the study of apoptosis, in order to understand, and eventually to manipulate this fundamental biological process.

Conclusion and future prospects of apoptosis research

Enormous progress has been made towards understanding the basic molecular mechanisms used by cells to kill themselves. Many questions still remain unanswered, and many apoptotic subprograms are still poorly understood, but new experimental models are constantly developed, and progress in the area of apoptosis is made very rapidly. Scientists have decrypted the main mechanisms of programmed cell death, and this has allowed them to develop apoptosis-related therapeutics which are now under clinical trial21. For example, the understanding of the molecular pathophysiology of neuronal apoptosis, which involves Bcl-2 proteins, the adapter protein Apaf-1, and homologues of the ced-9, -4, and -3 caspases, has allowed the development of therapeutics for Alzheimer's disease, Parkinson's disease, and many other progressive neurodegenerative disorders19, 22, 23. The elucidation of the mechanism apoptosis-suppression of cytokines will hopefully allow to improve cancer therapy24,25. According to Lewis3, and to Kolenko et al.26, a better understanding of the molecular mechanisms of apoptosis in response to chemo- and immunotherapeutic strategies will help to avoid ineffective treatment regimes and provide a molecular basis for new strategies targeting caspase-independent death pathways in apoptosis-resistant forms of cancer and for gene therapy treatments. The proper understanding of apoptosis can also help in therapeutic interventions against inflammation and inflammatory diseases such as rheumatoid arthritis27,28. According to Savill & Fadok28, the phagocytic clearance of cells dying by apoptosis causes phagocyte responses that modulate inflammation (by increasing [TGF-1] and [PGE2] and decreasing [TNF]), control tissue remodelling by phagocyte-directed cell-killing (increasing [CD95L] and decreasing [NO]), and regulate immune response (via class I and II MHC). It can also play an important role in the treatment of cardiovascular diseases29, of renal diseases30,31 , of sunburns, of uterine and intestinal infections, and of eye diseases31.

The examples mentioned above clearly show the importance of the study of apoptosis in almost all the fields of medicine. There is still a lot to be learned about apoptosis, and there are still many problems to overcome before apoptosis-based therapeutic agents can be used on a regular base in the clinic. Hopefully, reasearch in the area of apoptosis will remain as successful as it has been in the last decades. The pace of apoptosis research has, indeed, raised expectations that apoptosis-based therapeutics will soon become available in the clinic and on the shelf 32.

References

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Appendix

Abbreviations

AIF:

Apoptosis-inducing factor

BH:

Bcl-2 homology

CARD:

Caspase activation and recruitment domain

C. elegans:

Caernorhabditis elegans

Ced:

Cell death defective

CD95L:

CD95 ligand

DED:

Death-effector domain

D. melanogaster:

Drosophila melanogaster

FADD:

Fas-associated death protein domain

hid:

Head involution defective

IAP:

Inhibitor of apoptosis proteins

ICE:

Interleukin-1-converting enzyme

MHC:

Major histocompatibility complex

NO:

Nitric oxide

PGE2:

Prostaglandin E2

PTP:

Permeability transition pore

TGF-1:

Cytokine transforming growth factor

TNF:

Tumour necrosis factor

TUNEL:

TdT-mediated dUTP nicked labelling

VDAC:

Voltage-dependent anion channel

7-AAD:

7-amino-actinomycin-D