An adult human is made up of about 30 trillion (3 × 1013) cells, allof which originate from a single fertilized egg. If this first cell dividesinto 2, the progeny cells into 4, and so on, it would take only about 45 rounds of division to produce the number of cells required to make an adult human. In fact, cell division occurs constantly through our lifetimes, such that we generate a new complete set of 3 × 1013 cells every 2 weeks. The reason that multicellular organisms do not

become infinitely large is because the proliferation of cells is balanced by cell death.

*Necrosis was the earliest form of Cell Death known to man and was considered to be the only one until Virchow’s landmark article titled,” Cellular Pathology” talked about involvement of cells in Infecction.* Necrosis was mentioned in Ancient Greek texts since Galen’s time. ( AD-129 )*Three main avenues leading to cell death have been delineated: necrosis, apoptosis andautophagy.*Necrosis was defined as an accidental form of cell death caused by a hostile environment to which a cell could not adapt effectively. It was thus seen as a passive process in which the cell was more sinned against than sinning itself.*By contrast, apoptosis is a form of cellular suicide in which the cell participates actively in its own demise. It is a mechanism by which individual cells activate their own signaling systems to sacrifice themselves for the preservation of the organism.*Autophagy (see above) is also an active signaling process that is elicited when a stressful environment requires autodigestion of a portion of the cell’s macromolecular constituents.

Development of the Cell Death Concept

*Necrosis is a form of “pathological cell death”.*Unplanned murder of cells by external agents.*Necrosis was defined as an accidental form of cell death caused by a hostile environment to

which a cell could not adapt effectively. *It was thus seen as a passive process in which the cell was more sinned against than sinning

itself.*Typically affects geographically localized groups of cells. The response to this process is

usually acute inflammation, which itself may generate further cell injury.

In cells that are injured, ATP concentrations fall so low that the Na+/K+ ATPase can no longer operate, and therefore

ion concentrations are no longer controlled. This causes the cells to swell and then burst. The cell contents then leak

out, causing the surrounding tissues to become inflamed. Cells that die by suicide on the other hand shrink, and their

cell contents are packaged into small membrane-bound packets called blebs. The nuclear DNA becomes chopped up

into small fragments, each of which becomes enclosed in a portion of the nuclear envelope. The dying cell modifies its

plasma membrane, signaling to macrophages, which respond by engulfing the blebs and the remaining cell fragments .

*Three morphologic changes follow:■ Pyknosis: The nucleus becomes smaller and stains deeply basophilic as chromatin clumping continues.■ Karyorrhexis: The pyknotic nucleus breaks up into many smaller fragments

scattered about the cytoplasm.■ Karyolysis: The pyknotic nucleus may be extruded from the cell or it may

progressively lose chromatin staining.

Necrosis

Coagulative necrosis.A.Normal heart. All myocytes are nucleated, and striations are clear. B. Myocardial infarction. The heart from a patient Following acute myocardial infarction. The necrotic cells are deeply eosinophilic and most have lost their nuclei.

APOPTOSIS

BARE BEGINNINGS*Spontaneous cell death as a physiological event was discussed almost as soon as stains became available. It was born with a bang in 1885 in a paperby the same Walther Flemming who created the terms chromatin and mitosis. Flemming studied ovarian follicles in mammals and noticed that the epithelial lining of regressing follicles was littered with cells the nuclei of which were breaking up.

*Sydney Brenner's studies on animal development began in the late-1950s in what was to become the Laboratory of Molecular Biology (LMB) in Cambridge, UK. It was at this lab that during the 1970s and 1980s, a team led by John Sulston succeeded in tracing the nematode Caenorhabditis elegans entire embryonic cell lineage. In other words, Sulston and his team had traced where each and every cell in the roundworm's embryo came from during the division process, and where it ended up.

Apoptosis as observed in 1885 by Flemming, who called it chromatolysis. Top left: Normal rabbit ovarian follicle near matunity, 1 mm in diameter. Numerous epithelial mitoses. Top right: Early stage of involution in a nearbyfollicle. Many epithelial cells are in vanous stages of death by chromatolysis; some are shed into the lumen. Bottom: detail of the same involuting follicle. Most epithelial cells in contact with the ovum are normal (one is in mitosis); those farther removed are undergoing chromatolysis. Note the half-moons of chromatin typical of apoptosis. (Osmium fixation; safraninand gentian violet staining. Camera lucidadrawings.)

Apoptosis as seen in 1886 by a German medical student, Franz Nissen, in the lactating mammary gland. Nissen became aware of Flemming's study,2' after having completed his own, and concluded that the name chromatolysis was very suitable also to his own findings.

Apoptosis illustrated in 1914 by L. Graper as chromatolysis. Wall of the yolk sac in the course of involution, in a 20-cm embryo of Acanthias, a creature that we have been unable to identify. An epithelial cell has taken up the fragmented nucleus of a neighboring cell that died during the involution process.

Apoptosis actually

• What’s in a name?

• The term, ‘Apoptosis’ was coined in 1972 by Kerr, Wyllie and Currie in their landmark article introducing the concept of Programmed cell death titled, “APOPTOSIS: A BASIC BIOLOGICAL PHENOMENON WITH WIDE- RANGING IMPLICATIONS IN TISSUE KINETICS.”

• We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word " apoptosis " is used in Greek to describe "the dropping off " or " falling off " of petals from flowers, or leaves from trees. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like " ptosis " (with the " p " silent), which comes from the same root " to fall” and is already used to describe drooping of the upper eyelid.

• A general mechanism of controlled cell deletion, which is complementary to mitosis in the regulation of animal cell populations.

The two main regulators of cell populations, mitosis (bottom left) and apoptosis (top right). In the apoptotic cell, the nucleus is fragmented (karyorhexis) and the chromatin is pyknotic. One of the nuclear fragments contains a characteristic half-moon of condensedchromatin. (Electron micrograph from a rat prostate 2 days after castration. Bar = 2 Y).

*Apoptosis is a pattern of cell death that is triggered by a variety of extracellular and intracellular stimuli and is carried to its conclusion by organized cellular signaling cascades.*Apoptotic cells are recognized by nuclear fragmentation and pyknosis, generally Against background of viable cells. Importantly, apoptosis occurs in single cells or small groups of cells, whereas necrosis characteristically involves larger geographic areas of cell death. .

Apoptosis Produces Individual Cell Death Amidst

Viable Cells

The earliest phase is the stimulus that provokes the apoptotic response. This may be an external signal delivered through surface receptors or may originate inside the cell from the action of a drug, toxin, or radiation. The next phase includes detection of this signal or metabolic state and transduction of the signal. Signal transduction pathways send this message to the cell death effector machinery. The effector phase is the third part of the cell death mechanism and includes the proteases that are activated during apoptosis, as well as their positive and negative regulators.The fourth phase of cell death is the postmortem phase, in which the cell's chromatin condenses and its DNA is degraded. In vivo (but not necessarily in vitro) dying cells are recognized and engulfed by other cells.

Stages

When, where and why?*Programmed cell death rids the developing animal of superfluous or unwanted cells.*For example, large numbers of self-reactive lymphocytes orlymphocytes that fail to produce useful antigen-specificreceptors are deleted by programmed cell death.*In the developing nervous system, about half of the neuronsproduced undergo programmed cell death soon after they mature if they fail to establish vital connections with target.*Cells dangerous to the organism, such as transformedcells, damaged cells, or those infected with pathogens such as viruses, are also eliminated by the activation of programmed cell death.*In case of DNA Damage, PCD may eliminate cells carrying potentially harmful mutations, including mutations in cellsthat can lead to development of Cancer.

Removal of Apoptotic CellsOnce the self-destructive process of apoptosis has propelled a cell to DNA fragmentation and cytoskeletal dissolution, the final phase, the apoptotic body, remains. Apoptotic bodies are phagocytosed by tissue macrophages. Phosphatidylserine (PS), a phospholipid that is normally on the interior aspect of the cell membrane, is externalized in cells undergoing apoptosis. PS is recognized by macrophages and activates ingestion of an apoptotic cell’s mortal remains without release of intracellular constituents, thus avoiding an inflammatory reaction.Mononuclear phagocytes ingest the debris from apoptotic cells, but recruitment of neutrophils or lymphocytes is rare. This situation is unlike that of cells that undergo necrotic cell death, which tends to elicit acute inflammatory responses.

Molecular mechanism

*Insight into the molecular basis of apoptosis was first revealed in studies on the nematode worm C. elegans, whose cells can be followed with absolute precision during embryonic development. *Of the 1090 cells produced during the development of this worm,131 cells are normally destined to die by apoptosis yielding 959 somatic cells seen in adult worm. (Sulston and Horvitz, 1977; Sulston et al., 1983).

* Nearly all dying cells undergo the same sequence of morphological changes . (Sulston and Horvitz, 1977; Sulston et al., 1983).

*A number of mutations affecting the process of cell death have been isolated. These mutations, which have defined the genes ced-7 (for cell death abnormal), ced-2, and nut-7 (nuclease deficient) (Sulston, 1976; Hedgecock et al., 1983), affect all programmed cell deaths. Thus, all cell deaths appear to involve the same genetic (and, hence, molecular) processes.

Crucial Findings*In 1986,Robert Horvitz and his colleagues at the Massachusetts

Institute of Technology discovered that worms carrying a mutation in the CED-3

gene proceed through development without losing any of their cells to apoptosis.

This findingsuggested that the product of the CED-3 gene played a crucial role in

the process of apoptosis in this organism.*Two genes, CED-3 and CED-4, were identified as genes required for

cell death: Inactivating mutations in either of these two genes prevents all

developmentally occurring cell deaths in C. elegans. This observation also provided

the first direct evidence that cells die by an intrinsic suicide program. *CED-3 is a protease, whereas CED-4 is a protein that activates CED-

3 by interacting with it in dying cells. Another gene, CED-9,acts to protect cells from

programmed cell death.*CED-9 blocks death by interacting with CED-4, thus preventing it

from activating CED-3. Inactivating mutations of CED-9 lead to the death of cells that

would normally survive through development; thus, worms with CED-9 mutations die early.

*Complicating the issue, CED-9 is also expressed in many of the 131 cells

that are fated to die during development. How then do these cells die? Death of these cells requires the expression of another gene, EGL-1.

EGL-1 antagonizesCED-9 by physically interacting with it, thus freeing CED-4 to activate

CED-3.Genetic studies have shown that the over expression of EGL-1 or

mutations thathyper activate EGL-1 (gain of function mutation) can induce ectopic cell

death in C. elegans, which can be suppressed by gain-of-function mutation of CED-

9 or loss of function mutations of CED-3 or CED-4. *In contrast, a loss of function mutation of EGL-1 cannot suppress cell

deaths caused by the loss of CED-9 activity (loss of function CED-9). These results are

consistent with EGL-1 acting upstream of the CED-3, CED-4, and CED-9 genes in the

genetic pathway regulating cell death.

*The identification of key components of the genetic program in the nematode led to

the search for their homologs in mammals. These studies have resulted in the

identification of a family of proteins, collectively called caspases, as the mammalian

homologs of CED-3.*The homolog of CED-4 in mammals is a protein called Apaf-1,

whereas proteins related in sequence to CED-9 comprise the Bcl-2 family. In contrast

with the situationin C. elegans, however, where CED-9 is antiapoptotic, the Bcl-2 family

of proteins is composed of both antiapoptotic and proapoptotic members. Some of these

proapoptotic Bcl-2 proteins function as the mammalian counterparts of EGL-1. *Consistent with the evolutionary conservation of the mechanisms

underlying apoptosis, the overexpression of the nematode proteins CED-3 or

CED-4 in mammalian cells results in apoptosis,whereas overexpression of Bcl-2

can compensate for the lack of CED-9 in the nematode.

Isolation of Cell Death Mutants by Direct Screening

Programmed cell deaths can easily be identified in living nematode using Nomarski differential interference contrast microscopy (Sulston and Horvitz, 1977). The first sign of the impending death of a cell is a slight increase in its refractility. *The nucleus of the dying cell becomes increasingly refractile until it resembles a flat button; this stage persists for lo-30 min.*Subsequently, the nucleus of the dying cell decreases in refractility, begins to appear crumpled, and then gradually disappears. This process is completed in less than 1 hr .(Sulston and Horvitz, 1977;Sulston et al., 1983).

Ced-9 mutant (below)

Absence of Cell Deaths in ced-3 Animals

Ghgm;;

References*Cell and Molecular Biology- Gerard Karp.*Apoptosis, Oncosis, and Necrosis-Guido Majno and Isabelle Joris From the Department of Pathology, University of Massachusetts Medical School, Worcester,Massachusetts. American Journal of Pathology, Vol. 146, No. 1, January 1995 Copyright ) American Society for Investigative Pathology.

*Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue Kinetics.J. F. R. KERR*, A. H. WYLLIE AND A. R. CURRIE. Br. J. Cancer (1972) 26, 239

*Genetic Control of Programmed Cell Death in the Nematode C. elegans.Hilary M. E.llis,’ and H. Robert Horvitz.Cell. Vol 44, 817-829, March 28, 1986, Copyright 0 1986 by Cell Press.

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