Apoptosis: A Programmed Cell Death

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Rajiv Dahiya

Rajiv Dahiya

Cell death is currrently the subject of considerable research activity. This interest stems from the possibility of exploiting the cell death program for therapeutic purposes. Cell injury can be irreversible or reversible. Apoptosis (pronounced “ay-puh-TOE-sis”) is a reversible pathway of cellular “suicide” which usually takes place by two major routes viz. internal / external signals and death deactivators.

Moreover, apoptosome and caspases are the vital contributions of programmed cell death.  Regulation of this form of cell death comprises of participation of bcl-2 proteins, TP53 gene, nitric oxide as well as steroid hormones. In real sense, apoptosis involves deliberate elimination of cells occuring in a morphologically distinct manner that suggests an active, tightly controlled gene-directed process. Therefore, a single sentence definition for apoptosis comprises ‘gene-directed cellular self-destruction’.

Introduction

The health of multicellular organisms depends not only on the body's ability to produce new cells but also on controlled cell death. For every cell, there is time to live and a time to die. Cells may be either killed by injurious agents viz. toxic chemicals and mechanical damage or they are induced to commit suicide. Apoptosis (Greek:apo, off; ptosis , a falling – ‘falling of leaves’) 1,2 term was coined by Kerr in 1972 and was first described as a "shrinkage necrosis", and later on it was replaced by ‘apoptosis’ to emphasize its role ‘opposite mitosis’ in tissue kinetics. In real sense, apoptosis is a physiological cell death process by which single cells are removed from the midst of living tissue without disturbing its architecture or function, or eliciting an inflammatory response. The ordered pattern of events taking place in apoptosis can be summarized in following sequence:

  • Shrinkage of cells and development of bubble like blebs on cell surface
  • Degradation of chromatin (DNA and Protein) and breakdown of mitochondria with release of  cytochrome c inside nucleus of cells
  • Breakdown of the cell into small membrane wrapped fragments
  • Exposure of Phospholipid phosphatidylserine (PS) on the cell surface
  • Binding of exposed PS with receptors on the phagocytic cells (macrophages) which engulf  the  cell  fragments
  • Secretion of cytokines by Phagocytic cells and inhibition of inflammation  (cell death)

Why a cell commits suicide ?

There are two reasons for a cell to commit suicide:

1) Programmed cell death is needed for proper development.

      Examples :

  • The formation of fingers and toes of the fetus requires the removal of the tissues between them which occurs through apoptosis.
  • The sloughing off of the inner lining of the uterus at the start of menstruation occurs by   the apoptosis.
  • The formation of the proper connections between neurons in the brain requires elimination   of the surplus cells which occurs by apoptosis. 

2) Programmed cell death is required to destroy cells that represent a threat to the integrity of the organism.

      Examples :

  • Cells infected with viruses
  • Cells of the immune system
  • Cells with DNA damage
  •  Cancer cells

Mechanisms of Apoptosis 3~7

Normal cells die almost always by the process of apoptosis following well-ordered cell death program which mainly consists of four separable but overlapping components viz. signaling, control and integration, execution and removal of dead cells. There are two major pathways by which a cell commits suicide by apoptosis 8~11 :

1) By signals arising within the cell (Intrinsic Pathway)

2) By death deactivators such as Tumor Necrosis Factor-alpha (TNF-a) and Fas ligands (FasL) binding to receptors at the cell surface (Extrinsic Pathway).

Intrinsic Pathway :

Intrinsic signals initiate apoptosis by the involvement of mitochondria 12~14 which releases cytochrome c and thereby activating caspase 9 and caspase 3. This effect is mediated through formation of an apoptosome - a multi-protein complex consisting of cytochrome c, pro-caspase 9 and ATP. Caspases are Cysteine Aspartate Specific Proteases 15,16 that exist within the cell as inactive pro-forms of zymogens which can be cleaved to form active enzymes following the induction of apoptosis. During this pathway, apoptogenic effects develop in three phases:

a) Pre-mitochondrial :

This phase comprises interaction of apoptogenic signals such as genotoxic agents, oxygen free radicals 17 , corticoids and antibodies, with cell receptors.

b) Mitochondrial :

During this phase, mitochondrial damage results which include membrane permeabilization, formation of transmembrane pore across the outer mitochondrial membrane, collapse of the membrane potential, swelling, membrane disruption, inhibition of electron transfer and oxidative phosphorylation.

c) Post-mitochondrial :

During the third phase of apoptosis, free radicals and activated enzymes attack the cell protein structure and therefore, causing cell death.

Moreover, there may be involvement of apoptosis-inducing factors (AIF) which are normally located in the intermembrane space of the mitochondria. When the cell receives a signal telling it that it is time to die, AIF releases from the mitochondria, migrates into the nucleus, binds to DNA and finally triggers the destruction of DNA followed by cell death.

Extrinsic Pathway :

This pathway include involvement of death receptors 18 that rapidly induces apoptosis which comprises of signaling by:

  • TNF Receptor-alpha
  • Fas (CD95)
  • TRAIL (TNF-related apoptosis inducing ligands) 19

Apoptogenic effects by death receptor pathway develop by formation of caspase cascade occuring as follows:

a) Initiator Caspases :

Death receptors activate caspase 8 or caspase 10 which acts as initiator caspases that further activate another caspases in a cascade.

b) Effector Caspases :

This cascade eventually leads to the activation of the effector caspases such as caspase 3 or caspase 6 which are responsible for cleavage of the key cellular proteins that leads to the typical morphological changes observed in cells undergoing apoptosis 20 .

Furthermore, induction of apoptosis following UV exposure appears to be a protective mechanism. UV-mediated apoptosis 21 is a highly complex process which include different molecular pathways such as DNA damage, activation of the tumor suppressor gene TP53 .

Critical Regulators of Apoptosis

Bcl-2 Proteins :

Bcl-2 and genes of this family 22,23 are directly involved in regulation of release of cytochrome c which is believed to be the key event in apoptosis. These proteins are of two types :

1) Anti-apoptotic : These  proteins  inhibit  apoptosis  by  preventing  release of  cytochrome c.

 When  there  is  an  excess of anti-apoptotic proteins, the cells will tend to be less sensitive to  apoptosis. Examples :  Bcl-2, Bcl-xL.

2) Pro-apoptotic : These   proteins  promote  apoptosis  by  favouring  release  of  cytochrome c.

 When there is an excess of pro-apoptotic proteins, the  cells  are more sensitive to  apoptosis.  Examples :  Bad, Bax or Bid.

The sensitivity of cells to apoptotic stimuli can depend on the balance of pro- and anti-apoptotic Bcl-2 proteins. The interaction between the pro- and anti-apoptotic proteins disrupts the normal function  of  anti-apoptotic  Bcl-2  proteins  and  can  lead to formation of pores in mitochondria followed by release of cytochrome c and thus, regulating apoptois.

TP53 Gene :

TP53 ( p53 ) 24 tumor suppressor gene and cell growth associated gene c-myc 25 are involved in the regulation of apoptosis. TP53 senses DNA damage by unknown mechanisms and assists in DNA repair by causing G 1 arrest and inducing DNA repair genes. A cell with damaged DNA that can not be repaired is directed by TP53 to undergo apoptosis. In view of these activities, TP53 has been rightfully called a “ Guardian of the Genome . TP53 regulates apoptosis 26 but equally, it can exert antiproliferative effects.

Hormones :

Many hormones and growth factors can either prevent or induce apoptosis under physiological or pathological conditions.

1) Steroid hormones 27 :

These  are  potent regulators of apoptosis in steroid-dependent cell types  and tissues such as mammary gland, prostate, ovary and testis.

2) Growth factors : 

Epidermal   growth   factor,   nerve growth  factor,  platelet-derived growth      factor (PDGF) and insulin-like growth factor-I act as survival factors and inhibit apoptosis in   a  number of cell types such as haematopoietic cells, preovulatory follicles, mammary gland, phaeochromocytoma cells and neurons.

Conversely, apoptosis modulates the functioning and the functional integrity of many endocrine glands as well as many cells that are capable of synthesizing and secreting hormones.

Nitric oxide :

Nitric oxide 28 (NO) is a multifunctional signaling molecule that is synthesized by a family of enzymes, nitric oxide synthases (NOS). Depending on dose, NO can either induce or protect apoptosis 29,30 in different cell types. For instance, NO inhibits apoptosis in leukocytes, hepatocytes, trophoblasts and endothelial cells. The effects of NO on apoptosis are generally classified as cGMP-dependent 31 or independent. Anti-apoptotic effects of NO can be mediated through a number of mechanisms viz. nitrosylation and inactivation of caspases 1, 3 and 8. Besides it, NO is also implicated in several physiological functions within the microvascular environment 32 i.e. regulation of vascular tone, antiplatelet and antileukocyte properties and modulation of cell growth.

Significance

It has become clear that apoptosis plays an important role in organ and tissue development. Defects in apoptotic cell death program contribute to pathogenesis of cardiovascular diseases 33 such as heart failure, ischemic heart disease and cardiac hypotrophy, wide variety of skin diseases 34 , ocular diseases 35,36 , lung diseases 37 , autoimmune diseases 38,39 , atherosclerosis 40 and cancer 41~43 whereas excessive apoptosis is always associated with neurodegenerative disorders 44 and AIDS 45 . Therefore, elucidation of the role of apoptosis in these diseases may lead to new possibilities for treatment 46,47 .

Summary

Apoptosis is an integral part of animal as well as plant development and studies on this vital cell death led to the identification of a ‘central death switch’ i.e. apoptosome and ‘central executioners of apoptosis’ i.e. caspases, both of which play key role in process of programmed cell death. As disordered apoptosis process participates in the pathogenesis of various diseases and conditions, pharmacologic manipulation of this pathway is a novel therapeutic target. Understanding the molecular events that contribute to apoptosis enable a more rational approach towards novel anticancer strategies allowing development of molecules targeting new apoptotic signaling pathways and thus offers widespread scope for novel approaches to the treatment of neoplasms. 

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About Authors:

Rajiv Dahiya

Rajiv Dahiya

Assistant Professor, Department of Pharmaceutical Chemistry, Rajiv Academy for Pharmacy, Mathura - 281 001, UP (India), *has published 12 research papers and 5 review articles in journals of international/national repute and presented more than 25 scientific papers in various international and national conferences.

Devender Pathak

Devender Pathak

Director, Rajiv Academy for Pharmacy, Mathura - 281 001, UP, India.

E-mail: dev_15@rediffmail.com, Phone: 09997036162 (Mob), 0565-2900385 (Off), 0565-329553 (Res) , Fax: 0565-2530766.

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