21spring 2011

The Role of Apoptosis in Disease and Development

Jay dalton ‘12

cell BiologY

I recently saw the Rude Mechanicals’ phenomenal adaptation of Hamlet, and for one reason or another, as

the final act drew to a close, I was in a decidedly morbid state of mind. Per-haps the acting prowess demonstrated by the troupe caused my suspension of disbelief to be utterly complete even in the prosaic setting of Brace Commons. Perhaps it was the sight of so many fa-miliar Dartmouth faces twisted in the throes of Shakespearean tragedy. Or, perhaps, it was simply because liter-ally all of the important characters in the play die. Regardless of the rea-son, the contrast between Ophelia’s tastefully understated offstage suicide and Hamlet’s melodramatic onstage murder got me thinking about the two vastly different cellular fates: apoptosis and necrosis. Necrosis, which is akin to Hamlet’s rapier-induced demise, is simply premature cell death, and can lead to the death of the organism in multi-cellular life if the damage is se-vere enough. Apoptosis, on the other hand, can be seen as the cell willfully shuffling off its mortal coil, along with its mortal condensed chromatin, apop-totic bodies, and other cellular mate-rial. The dramatic irony of apopto-sis, at least from the perspective of a researcher, is that the programmed death of the individual cell is essential to life processes ranging from embry-onic development, everyday organ sys-tem function, and bodily maintenance.

First described in 1842 by Carl Vogt, apoptosis was not precisely de-fined as programmed cell death until anatomist Walther Flemming’s work in 1885. It was not revitalized as a sub-ject of study until the middle of the 20th century (1). As it is understood today, apoptosis is initiated by two different means; either through targeting mi-tochondria functionality, or by using adaptor proteins to directly transduce the apoptotic signal. A multitude of environmental signals, which includes heat, radiation, nutrient deprivation, viral infection and hypoxia, has been

implicated in causing apoptosis (2,3).

Mechanism of Action Mitochondria-focused apoptosis

is further subdivided into two separate mechanisms (4). The first method of mitochondrial apoptosis is mediated by the formation of channels such as the mitochondrial apoptosis-induced channel (MAC) (4). MACs are ion pores formed on the outer mitochondrial membrane in response to apoptotic stimuli such as Bax and Bak, which are members of the Bcl-2 protein family that contains both pro- and anti-apop-totic signal carriers (4). Once induced, MACs release cytochrome c into the cytosol, which initiates the so-called commitment step of the mitochondrial apoptotic cascade (5). In fact, this path-way has been utilized therapeutically via Bax inhibitors in order to knock down MAC channels and thereby pre-vent this form of apoptosis (5). When cytochrome c is released, however, it binds with both apoptotic protease ac-tivating factor-1 and ATP, which then bind to pro-caspase-9 to create a pro-tein complex known as an apoptosome (4). This structure cleaves the pro-cas-pase to its active form, caspase-9, which in turn activates the effector caspase-3 (4,5). This type of signaling cascade is typical of biological signal trans-duction, and especially of apoptosis.

The other mitochondrial-apop-tosis pathway releases second mito-chondria-derived activator of caspases (SMACs) into the cytosol via an in-crease in mitochondrial permeability. SMACs bind to and inhibit inhibitor of apoptosis proteins (IAPs) (6). IAPs function normally by suppressing cas-pases, which are cysteine proteases (6). When IAP inhibition is lifted, caspases carry out the degradation of the cell (6).

The direct signal transduction method is less well understood. Two current theories implicate tumor ne-crosis factor (TNF) as the driving force behind one pathway and the Fas ligand

as the instigator behind the other (7). The result of cytokine production, TNF has two receptors in the human body: TNF-R1 and TNF-R2 (7). Binding of TNF to these receptors leads to cas-pase activation. The other direct signal transduction method, which utilizes the Fas ligand, is closely related to the TNF pathway. Binding of the Fas ligand to the Fas receptor results in the formation of the death-inducing signaling complex (8). Like most apoptotic targets, this complex contains a number of caspases.

Apoptosis in Normal Development

Programmed cell death might be the body’s best option compared to necrosis, but the link between apop-tosis and the generation of life is not exactly intuitive. Apoptosis is one of the key mechanisms of embryonic de-velopment of organs and structures in both humans and other animals, and is as much a part of embryo develop-ment as is cell proliferation and dif-ferentiation (8). In fact, apoptosis can occur even in the follicular stage and is greatly enhanced by androgen and gonadotropin releasing hormones. For instance, follicular atresia, the main process of the menstrual cycle, is pre-

Image retrieved from http://upload.wikimedia.org/wikipedia/commons/5/5f/Apoptosis_stained.jpg (Accessed 10 May 2011).

In this section of a mouse liver, a stained apoptotic cell is visible.

Dartmouth unDergraDuate Journal of science22

dominantly dependent on granulosa cell apoptosis (8). Currently, five cell-death ligand-receptor systems have been identified in granulosa cells that regulate atresia, all belonging to the TNF family (9). These receptors, upon contact with their respective ligand, form trimers that fit into the grooves formed between TNF monomers. As a response to this binding, a conforma-tional change occurs in the receptor, and in turn, the inhibitory protein, “silencer of death domains,” is disso-ciated (9). When dissociation is com-plete, the adaptor protein, “TNF recep-tor associated death domain protein,” is able to bind to the death domain (9).

A small proportion of follicles can escape initial apoptosis through/with the protection of growth factors and estrogens. However, programmed cell death occurs continually through-out embryogenesis. For instance, early brain development involves both Jnk1 and Jnk2 protein kinases, both of which are implicated in apoptosis. In fact, mutant mice lacking these proteins are embryonic lethal and were found to have severe apoptotic dysregulation. Additionally, normal fetal lung devel-opment is associated with a progres-sive increase of epithelial and intersti-tial apoptotic activity. In addition, from birth onwards, the number of cells un-dergoing apoptosis increases dramati-cally and occurs in a spatially, tempo-rally, and cell-specific manner. (10)

Contrarily, pregnancies with abnormally increased occurrence of apoptosis and apoptotic cells have the potential to impair the functions of the fetal membrane. This is the case in diseases such as fetal growth restric-tion (FGR). Although apoptosis oc-curs in both normal and FGR-affected fetal membranes, apoptotic cells are present in greater quantities and are concentrated in the chorionic tropho-blast layer of the FGR-affected fetal membrane. The chorionic trophoblast layer, which separates the mother from the developing fetus, is vital for nor-mal fetal development and growth, and increased apoptosis may impair this layer’s functions and lead to pre-mature rupture of the membrane. (10)

Beyond its role in the development of the fetal membrane, apoptosis is even involved in crafting such human char-acteristics as the spaces between our

digits during development (1). Apop-tosis also seems to play a crucial role in Thymocyte (T-cell) biology. T-cells ex-pressing nonfunctional or auto-reactive T-cell receptors are eliminated during development by apoptosis. Dysregula-tion of apoptosis in the immune system thus results in autoimmunity, tumoro-genesis, and immunodeficiency (4,5).

Role of Apoptosis in Disease, Inhibition and Excess

There is a wide range of diseases that can result from loss of control of cell death (excess apoptosis). These include neurodegenerative diseases, hematologic diseases, and general tis-sue damage. For example, the progres-sion of HIV is directly linked to ram-pant apoptosis. In a normal individual, CD4+ lymphocytes are in equilibrium with cells produced by bone marrow.However, in HIV-positive patients, this balance is lost due to the bone mar-row’s inability to regenerate CD4+ cells. When stimulated, CD4+ lymphocytes die at an accelerated rate through un-controlled apoptosis induced by HIV. In fact, this uncontrolled cell death is largely responsible for the progression of the human immunodeficiency vi-rus infection into AIDS due to the de-pletion of CD4+ T-helper lymphocytes. This leads to a compromised immune system and symptoms typical of AIDS. In addition, cells may die as a direct consequence of viral infection. HIV-1 expression induces tubular cell G2/M arrest and apoptosis (1,3). Similarly, Ascoviruses perform viral infection and replication through the induction of apoptosis. Cell fragmentation occurs upon the viral instigation of apoptosis, and it is postulated that the virus utiliz-es the apoptotic bodies in order to form vesicles (11). Viruses can remain intact from apoptosis particularly in the lat-ter stages of infection. Ascoviruses can be exported in apoptotic bodies that pinch off through the normal apop-totic procedure from the surface of the dying cell (11). Viral phages in these apoptotic bodies are then consumed by phagocytes, which prevent the ini-tiation of a host immune response (11).

Interestingly, viruses are able to

induce disease not only through in-creasing apoptosis, but also through inhibiting it. A significant component of an organism’s ability to stave off extensive bodily infection is through apoptosis. However, certain viruses can halt this process in order to pro-ceed with their invasion. Ordinarily, vi-ral infection induces apoptosis, either directly or through the host’s immune response. Accordingly, the purpose of the cell death is to reduce production of new virus and further cell infection. However, new inhibitory mechanisms have evolved in many viruses that thwart the completion of defensive cell destruction. Viruses utilize a range of mechanisms to inhibit apoptosis, in-cluding activation of protein kinase R (PKR), interaction with tumor sup-pressor p53, and expression of viral proteins coupled to major histocom-patibility complex (MHC) proteins on the surface of the infected cell (1,4).

An example of such inhibitory mechanisms is a quality of the Herpes Simplex Virus (HSV), which inhibits apoptosis through the action of two genes, us5 and us3 (11). HSV enacts a latent infection with quiescent, persis-tent qualities. In fact, it is the charac-teristic latent period of the virus that precipitates the inhibition of apoptosis mechanisms. Specifically, during latent infections of the cell the virus expresses Latency Associated Transcript (LAT) RNA. This form of RNA has the capabil-ity to regulate the host cell genome and interfere with cell death mechanisms. LAT expression of the HHV-8 form of the virus produces miRNAs, which suppresses production of Thrombos-pondin-1 protein involved in apoptosis and angiogenesis. Inhibitory peptides and fragments of TSP1 bind to Clus-ter of Differentiation 36 (CD36), an

Image courtesy of CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

HIV-1 virions present on a human lymphocyte.

23spring 2011

integral membrane protein, leading to lowered expression of the FAS ligand, which normally activates the expres-sion of Fas-receptor induced apoptosis. Additionally, expression of LAT by an HSV virus reduces the production of other proteins involved in the apop-tosis mechanism, including proteins caspase-8 and caspase-9. Through the inhibition of apoptosis mechanisms during LAT expression, host cells are maintained and the proliferation of the virus is ensured (12). Although these cells would normally die to prevent outbreaks, the virus has effectively dis-abled the cells’ apoptotic abilities and has created a habitable environment for dormancy and further infection.

In addition to viral infections, apoptotic inhibition can result in a number of cancers, autoimmune dis-eases, and inflammatory diseases. The most logical result of a lack of pro-grammed cell death is cancer, which is usually characterized by an over-expression of IAP family members (1,4). As a result, malignant cells expe-rience an abnormal response to apop-tosis induction: cycle regulating genes,

such as p53, ras or c-myc, are mutated or inactivated in diseased cells, and other genes, such as bcl-2, also mod-ify their expression in tumors (15).

ConclusionIt is clear that apoptosis is not

only a crucial biological process from pre-birth until death, but also a prom-ising and daunting target for disease research. From the spaces between our fingers to the army of immune cells that protect us, the ability to cause cells to die is as essential as the processes that keep them alive. A better under-standing of apoptosis could hold enor-mous promise in treating cancer, viral infections, and autoimmune diseases. More importantly perhaps, the full pic-ture of apoptosis could solve essential mysteries about how our physical be-ings are formed by elucidating the ar-chitecture and blueprints of embryonic development. All of these mysteries and possible advances are wrapped up in the cascade of signals holding each of our cells on the precipice of destruction at the hands of apoptosis.

References

1. J. Kerr, J. Pathol. Bacteriol. 90, 419-435 (1965). 2. S. Popov, Biochem Bioph. Res. Co. 293, 349-355 (2002). 3. B. Brune, Cell Death Differ. 8, 864-869 (2003). 4. R. Cotran, K. Vinay, T. Collins, Pathologic Basis of Disease (Philadelphia: W.B Saunders Company Thomson, 1998).5. S. Fesik, Y. Shi, Science. 5546, 1477-1478, (2001). 6. L. Dejean, S. Martinex-Caballero, K. Kinanally, Cell Death Differ. 8, 1387-1395. (2006). 7. G. Chen, D. Goeddel, Science. 5573, 1634-1635. (2002). 8. H. Wajant. Science. 5573, 1635-1636. (2002).9. S. Santos et al., J. Cell Biol. 192, 571-580. (2000). 10. C. Haanen, I. Vermes, Eur. J. Obstet. Gyn. R. B. 1, 129-133 (1996). 11. M. Hussain, S. Asgari, Apoptosis. 12, 1417-1426 (2008). 12. A. Gupta et al., Nature. 7098, 82-85 (2006). 13. A. Rolaki et al., Reprod. Biomed. Online. 1, 93-103 (2005). 14. L Old, Science. 4726, 630-2 (1985).15. P. Murthl et al., Placenta. 4, 329-338 (2005). 16. K. Jerome et al., J.Virol. 73, 8950-8957 (1999).