Indian J Physiol Pharmacol 1995; 39(2): 91-94

REVIEW ARTICLE

APOPTOSIS IN HEALTH AND DISEASE

NEETA SINGH* AND SHUBHA ANAND

Department of Biochemistry,All India Institute of Medical Sciences,New Delhi - 110 029

( Received on May 10, 1994 )

Ahstract: Two distinct forms of ccll dcath are known, necrosis which results,from physical or chemical insult and apoptosis or programmed cell death resultsfrom programming within thc cell for self destruction ir. response to internal andexternal stimuli. Apoptosis is a genetically governcd process of ccll dcathoccurring in .dcvclopment and maintenance of multicellular organisms. It occllrsto get rid of individual cells that hecome unwanted for various reasons or Lhatpresent a threat to the organism. It is accompanied hy distinct morphologicalchanges. DNA fragmentation ,in most cases, and appears Lo be caused hy thcactivities of specific genes. Its defective regulation may play a part in Lheaetiology of cancer, AIDS, autoimmune and degenerative diseases. Apoptosisoffers potential for prevention and therapeutic modulation of these disorders.

Key words: apoptosis cell death genes cancer

INTRODUCTION

EukarYQtic cells die by one of the two stereotypicprocesses, necrosi's or apoptosis. In necrosis the cell iskilled by the hostile microenvironment whereas inapoptosis the cell is activated by specific signals tocommit suicide in a normal environment. Apoptosis awidespread biological phenomenon is a geneticallygoverned active process of cell death. The cell deathappears to be fixed developmentally and is thusprogrammed, and hence also called programmed celldeath. A multicellular organism must keep a balancebetween proliferation and cell death to maintain theproper number of cells. Thus during development alarge number of cells die by apoptosis. In fact theoriginal notion of apoptosis came from embryonicdevelopment studies.

Cell death occurs during normal development andhomeostasis because the cells become unwanted forvarious reasons: I) cells that have been evolutionaryvestiges and seem to have no function e.g. interdigitcell death has been altered repeatedly in evolution tohelp adapt the shape of the foot to newmicroenvironment, 2) cells generated in excess e.g.many vertebrate neurons; sometimes these excess cellsprovide the organism with devel.opmental plasticity, 3)cells that develop improperly e.g. in visual system of

*Corresponding Author

developing vertebrate, cell death preferentiallyeliminates neurons with improper connections, 4) cellsthat have already functioned e.g. resorption of tai I oftadpole and 5) cells that are harmful and must beeliminated to protect the animal e.g. death of immatureT cells in mouse thymus. An organism must <1lsoremove senescent, damaged or abnormal cells thatcould interfere with organ function or develop intotumors. There is potential significance of apoptosisbecause of its involvement in embryogenesis,differentiation, aging, metamorphosis, neuraldevelopment, epithelial turn.over like that of the skinand gut lining, tumor regression etc. Apoptosis is alsoobserved on withdrawal of trophic hormone from itstarget tissues and also in atrophy of prostate aftercastration. Cytotoxic effects of gl,uCOCOJ'ticoicis onimmature thymocytes and of tumor necrosis factor(TNF-a) on susceptible malignant cells, are also dueto apoptosis (I).

Abnormalities in the regulation of cell death maycontribute to the pathogenesis of both degenerativeand neoplastic disease. Horvitz (2) was firs! to showapoptosis ill vivo, even if it is in the wormCaenorhabditis elegans. He showed that out of the1090 cells in the worm, Dl cells die apoptotically asthe worm sculpts itself to maturity and that activaLionof cell death genes ced -3 and ced -4 seemed necessary

92 Singh and Anand Indian J Physiol Pharmacol 1995; 39(2)

for apoptosis whereas ced -9 was suppressor ofapoptosis.

Three main sets of events have been described inthe dying apoptotic cell: I) distinct morphologicalchanges like cell shrinkage, compactation of thechromatin, early collapse of nucleus, nuclear andcytoplasmic budding to form membrane boundapoptotic bodies, which are rapidly phagocytosed byadjacent cells or macrophages (3). Ingestion ofapoptotic cells by macrophages does not re'lease toxiccontents into extracellular space, and unlike necrosisis not accompained by inflammatory response, thusdoes not damage the adjacent ceUs, 2) The molecular/biochemical events involve fragmentation of nuclearDNA into 180 base pair multiples corresponding tothe size of a nucleosome. Most reports suggest theinvolvement of a Ca2+ Mg2+ endonuclease in thiscleavage response (4), 3) There is no uniform sequenceof metabolic events that occur during apoptosis, butmany upstream and downstream molecules have beenimplicated such as receptor binding andphosphorylation. In most cell types, an early increasein cytosolic ionized calcium is noted (5) which in turncan activate the nuclear endonuclease that cleavesDNA, and/or a transglutaminase that crosslinkscytosolic proteins (6). Calcium dependent proteaseslike calpain may also degrade the cytoskeleton.Apoptosis can often be inhibited or delayed by RNAsynthesis inhibitors like actinomycin D, or proteinsynthesis inhibitors like cycloheximide (7). It dependson the cell type in question and the apoptotic stimulus.Thus most cells contain regulatory protein that caneither inhibit or promote apoptosis. A variety ofapoptotic modulators have been described in differentsystems. They include zinc ion, calcium chelators,agents that stimulate protein kinase C e.g. phorbolester PMA, or interfere with calcium/calmodulinpathway, protein and RNA systhesis blockers, drugsthat inhibit poly ADPR polymerase i.e. 3 amino­benzamide, polyamines, and cyclosporin (8).

Apoptosis and cancer:

Many carcinogens damage DNA and the cell in turnresponds to this insult by either repairing the damageprior to replicating, or progresses through cell growthcycle, or undergoes apoptosis. Thus cells death byapoptosis prevents malignant transformation whereasabnormal apoptosis can promote cancer development.We still do not know what factors determine as to whichof the above pathways are to be followed by the cell.

Cell death by apoptosis is an active process andappears to be caused by the activities of pecificgenes that act within the dying cells. The productof these genes might be synthesized only after acell has been determined to die or alternativelythese products though present, become activatedonly in cells undergoing apoptosis. Interestinglysome of the immediate early genes that stimulateproliferation such as c-myc, c-fos and c-jun alsoappear to be increased in apoptosis (9). Theirovcrexpression induces apoptosis in fibr·oblasts.Many human cancers have mutation or deletionin the p53 gene. This gene is not only an inhibitorof cell division but also a mediator of apoptosis (10).Among the suppressor gene for apoptosis is the bcl-2oncogene. Hemopoietic, lymphoid cell's, many epithelialcells and neurons contain bcl-2. Follicular B-celllymphocytes that overexpress bcl-2 have a longersurvival in culture on growth factor deprivation andare also resistant to radiation and glucocorticoid inducedapoptotic cell death. Epstein Barr virus proteins increasethe expression of bcl-2 in Burkins lymphoma cells(II). Many different categories of diversechemotherapeutic agents such as topoisomeraseinhibitors, antimetabolites, alkyllating agents, and'hormone antagonists kill sensitive cells by apoptosis(12). Thus antitumour approaches need to be re­examined in the light of apoptosis.

Apoptosis and the immune system :

Cell death is an important component in the normalfunctioning of the immune system (13, 14). Theimmune system also appears to sculpt its antigendefending system by first building a large T cellpopulation and then selectively killing only autoreactiveT cells by clonal selection, a situation similar toembryonic development. The cdls seem Ito die silentlywithout provoking any kind of immune response. Thisis because the T cell receptor can recognize self­antigens present within the mouse and if these cellsare allowed to survive and proliferate, an autoimmunedisorder could develop. /n vitro the death of entirepopulation of immature thymocytes can be inducedwith glucocorticoids, radiation etc. (15). 8 lymphocyteshave also been shown to undergo apoplosis but moststudies have been performed on T lymphocytes and T­cells hybridomas.

TNF-a, a 17 KDa protein triggers apoptosis bybinding to its receptor. It is predominantly secreted by

Indian J Physiol Pharmacol 1995; 39(2) Cell Death by Apoptosis 93

activated macrophages 06). We studied the molecularmechanism of cell killing induced by TNF-a in thesensitive and resistant variants of mouse epidermalJB6 cells. Morphological and biochemica] changescharacteristic of apoptosis were found to precede TNF-ainduced cell death in TNF-a sensitive but not TNF-aresistant cells. The findings suggested that TNF-ainduced cell killing via apoptosis in TNF sensitivecells and that a preferential and transient ,increase in c­Jun dephosphorylation and AP-l transcriptional activitymight contribute to the preferential apoptotic responsein these cells (17). CTL mediated DNA fragmentationis similar to that described for developmental or TNF­a induced cell death, except the kinetics of itsappearance is very fast, as early as 5 min after addingCTL to their targets (18). The antiviral role of cytotoxicT cells might include the destruction of the virus in thehost cell by fragmentation of viral DNA (19). Someeffector T cells might destroy viruses within targetcells, through limited nucleic acid fragmentation, evenwithout target cell lysis (20) and no requirement fornew macromolecular synthesis. Senescence of neutrophilpolymorphs following deprivation of growth factors alsoinvolve apoptosis (21, 22).

Apoptosis appears to play a role in autoimmunediseases. Autoreactive T & B lymphocytes can begenerated throughout the life span due to random gellerecombination and mutation but normaJily, immaturelymphocytes that bind autoantigens die by apoptosis(23). However, defective deletion of these lymphocytesby apoptosis could predispose to autoimmunity. Fasantigen is a surface protein having homology withTNF-a cfnd nerve growth factor receptor and mediatesapoptosis (24). It is expressed in various human cells,including myeloid cells, T lymphoblastoid cells, diploidfibroblasts, in thymus, ovary, liver and heart (25).Mice carrying the lymphoproliferative mutation (I pr)have defects in Fas antigen gene. The 1pr seems tointerfere with T cell maturation and leads tolymphadenopathy with systemic lupus type ofautoimmune disease, indicating an important role forFas antigen in the negative selection of autoreactive Tcells. Mouse anti Fas monoclonal antibody has cytolyticactivity on human cells that express the antigen (26).Mouse T cell lymphoma transformed with human Fasantigen eDNA were killed by the anti Fas antibody byapoptosis.

Apoptosis ill AIDS:

Apoptosis has been suggested to have evolved as

antiviral defense (27). A kind of pathological imbalancebetween rate of CD4 cell death and cell replacementhas been seen in AIDS (28). Depletion of CD4 Tlymphocytes leads to immunodeficiency in HIV-linfected persons. It has been suggested that bothqualitative and quantitative defects in CD4 T cells inpatients with HIV infection may be the result ofactivation induced cell death or apoptosis (29). Thereis speculation that cross linking of CD4, molecules toone another by gp 120 alone or gp 120 in complexwith anti gp 120 antibodies prepares the cell forprogrammed cell death that occurs when an MHCclass II molecule in complex with antigen binds to theT cell antigen receptor. Thus, the mere activation of apreparatory cell by a specific antigen or superantigencould lead to the death of the cell, without directinfection by HIV. The apoptosis hypothesis wouldhelp explain the deilletion of CD4 T cells withoutrequiring that each depleted cell be infected withHIV-I. AIDS patients may have high levels of antibodyto APO- 1/ Fas and it is a factor in T cell death. Thusapoptosis offers potential in AIDS therapy, the onsetof immunodeficiency can be delayed by blocking animportant/essential step of cell death by apoptosis.

Apoptosis ill degenerative disease :

The removal of trophic factors and excessiveexposure to excitatory' amino acid neurotransmittersare toxic to neurons. On deprivation of nerve growthfactor, the sympathetic neurons undergo apoptosis, butthe high levels of bcl -2 in these neurons preventsapoptosis (30). Dying neurons have an apoptoticpathway that often requires RNA and protein synthesis.The exact role of apoptosis in Alzheimer's disease isunknown but it is suggested that the deposition of Bamyloid protein in the brain might prevent the neuronsfrom receiving the trophic factors or make-them moresusceptible to the cytotoxic effects of excitatory aminoacids.

Apoptosis provides an additional means ofregulating ceU number and so also biological activityand unlike simple degeneration, death that is dependenton active participation of cellular components can bepartiaHy suppressed. Elucidation of the mechanism ofapoptosis should provide new targets of therapy andhelp to design new drugs to certain degenerativediseases or to preventing death of acutely stressedceLLs or preventing death in cells undergoing the kindof long term stress as in Alzheimer's disease.

94 Singh and Anand Indian J Physiol Pharrnacol 1995: 39(2)

Apoptosis in therapy:Pharmacological manipulation of apoptosis will

make it possible to regulate the aging process andother diseases by increasing or decreasing thesusceptibility of particular cells to apoptosis. Apoptosisshould prove a new approach to chemotherapy ofepithelial cancers and to design new dmgs to somedegenerative diseases. Exploration of the alternativeidea that tumor cells could be eliminated by artificiallytriggering death through apoptosis seems attractive.

Drugs that promote apoptosis could amplify the effectsat' cancer chemotherapeutic agents on resistant cellsfor e.g. by inhibiting the expression of bcl-2. Inhibitorsot' apoptosis could be useful for AIDS, to treat diseasescaused by too much cel.l death e.g. Parkinson'~ diseaseor diseases with long term stress such as Alzheimer'sdisease. Gene therapy could DC beneficial todegenerative diseases. Apoptosis advocates hope toconquer cancer besides, ischemia, stroke, heart diseaseand autoimmunity in the near future.

REFERENCES

Fawthrop DJ, Hoobis AR. Davies DS. Mechanisms of cclldeath. Arch Toxic,,1 1991: 65:437-444.

2. Ellis RE. Yuan J. Horvitz RH. Mechanisms and functions ofcell dcath. Annrt Rev Cell Bioi 1991;7:663-698.

3. Kerr JF. Wylie AR. Apoptosis: a basic biological phenomcnonwith widc-ranging implications in tissue kinetics. lJr J Cancer1972; 26: 239-257.

4. Arends MJ. ~'Iorris RG. Wyllie AH. Apoptosis: The role ofthc endonuclcasc. /\m .I l'a/lu"1 1990: 136: 593-608.

5. Orrcnius S. McConkey DJ, Rcllomo G, NicOlc"a P. Rolc ofCa" in toxic cell killing. Trends Pharmacol Sci 1989: 10:281-285.

6. Knight CR. Rees RC. Griffin M. Apoptosis: a potential' rolcfor cytosolic transglutaminasc and ils importance in tumorprogrcssion. l3ioclJim 13iol'hys ACia 199 I: 1096: 312-318.

7. Thakkar NS. Abrogation of adriamycin toxicity il/ vivo bycyclohcxamide. Riochem Phamu/('ol 1992; 43: 16:';3-1691.

8. Bertrand R. Sobry E. Jcnkins 1. Pommier Y. Apoptosis andIts modulation in human promyelocytic HL-60 cells treatedwith DNA Topoisomerase I and II inhibitors. E.\P Cell Res1993: 207: 388-397

9. Oren M. The involvemcnt of oncogenes and tumor suppressorgenes in the control of apoptosis. Clincel' Mellis/lisis Rev1')90: II. 141-14:';.

10. Lowc SW. Sehimtl EM. Smith SW, Osborne 13A. Jacks T.P53 is rcquired for radiation-induccd apoptosis in mousethymocytes. Nil 11m: 1993: 362: 847-852.

I I. Finke J. Firtzen R. Ternes P Expression of bcl-2 in Burkitt',Iymphoilla cell lines: induction by latent Epstein-Barr virusgcncs. IJlood 1992: 80: 459-469.

12. Harry MA. Behnke CA, Eastman A. Activatioll ofprogrammed cell death (apoptosis) by cisplatin. otheranticancer drugs. toxins and hyperthermia. BiochelllF'III/III 1£/({I I 1990:40 2353-2362.

13. Cohen J1. Programmed cell death in the immune system..<ldv 111I111111/(1/ 1991: 50: 55-:';5.

14. Shwarl/. LM. Smith SW. Jones MEE. Osborne BA. Do allprogramilled cell deaths occur via apoptosis') 1'1"0(' Nil/II\clld Sci 1993: 90: 980-984.

l.~. Wyllie 1\1-1. Glucocorticoid-induecd thymocyte apoptosis i,assoc"lIed with endogcnous endonuclease aetivatioo. NaluI'e19811: 284: 555-5'\6

16. Wright SC, Kunlar P. Tan AW. Larrick JW. Apoptosis andDNA fragnlentation prceede TNF·(l induced cytolysis in U937 cells .I Cell fI;ochelll 1992: 48: 344-355.

17. Singh N. Sun Y. Colburn N. Molecular mechanism of putativeapoptosis induced by TNF-Ct. in RTIOI cells. Pro(' ;\m

Assocn Callcer Res 1993; 34 96.18. Cohen JJ, Duke RC, Chervenak R. DNA fragmentation in

targets of CTL: an example of programmed cell death in theimmune system. Adv I,'x/, Med Bioi 1985; 184: 493-508.

19. Clousten WII. Kerr JF. Apoptosis. Iymphocytotoxieity andthe eontaioment of viral production. ,"'ediclil Hypolhesis1985: 18: 399-404.

20. Martz E. Howell DM: CTL Virus eontrol cells first andcytolytic cells second') DNA fragmcntation. apoptosis andthe prelytie halt hypothesis. Immllnol Toda)' 1989: 10:79-:';6

21. Duke RC, Cohen JJ. IL-2 addiction: Withdrawal of growthfaclor activates a suicide programme in dependent T cell,.Lym/,Iwkine Re.( 1986: 5: 289-299.

22. Metcalf D. The molecular control of cell division.differentiation commitmcnt and maturation in haemopoietiecells. Nalure 1989; 339: 27-30.

23. Cohen JJ. Apoptosis and programmed cell death inl immunity.A/lIll1 Rev Immllno! 1992; 10: 267-293.

24. Itoh N, Yonehara S, Ishii A, Nagata S. 1i'he polypeptideencoded by the cDNA for human cell surface antigen Fascan mediate apoptosis. 1991: 66: 233-243.

25. Itoh N. Nagata S. A novel protien domain r;quired forapoptosis. Mutational analysis of human Fas antigen . .I lJiolChon 1993: 268: 10932-10937

26. Watanabe FR, Brannan Cl. Copel NG. Jenkins NA, NagataS Lymphoprolilcration disordcr in mice explained by defe.ctsin Fas antigen that mediates apoptosis. tVa/lire 1992: 365:314-317.

27. Laurent-Crawford AG. Krust 13. Muller S. Tbe cytopathiceffect of HIV is associated with apoptosis. Vil·olo.~y 1991;185: 829-839.

28. Pantalco G. Graziosi C. Fanci AS. The immunopathogenesof human immunodeficiency virus infection. tV EIli{/ .J J'vled

199.1: 328 327-335.29. Groux H, Topicr G. Monte D. Amcr,en Jc. Activation

induced cell death by apoptosis in CD4 T cells from humanimmunodcficiency vnus-infectcd asymptomatic individualsE.\II Med 1992: L75 331-340.

3D. Gracia I, Maninou I, Tsujimoto Y, Maninou Jc. Prcventionof rrogrammed cell death of syllnr'llhClic neurons I1y Ihc bel,2 protlJoncogene . .),·iel/ce 1992: 258: ,1D2-:\()4.