Apoptosis-A cell suicide

INTRODUCTION: Apoptosis is an active, programmed process of autonomous cellular dismantling, that avoids eliciting inflammation. (Or) Apoptosis is a form of programmed cell death or cell suicide mechanism where cell plays an active role in its own execution. Programmed cell death is defined as cell death that is dependent on genetically encoded signals or activities within the dying cell Pronounce: Apoptosis as Apo-ptosis not as Apop-tosis. Apoptosis in greek falling of leaves or petals. Apoptosis was distinguished from traumatic cell death by Jhon Foxton Ross Kerr (Emeritus professor of pathology at the university of queensland) Kerr wylie and curie credited professor James Cormack (Department of greek ,University of Aberdeen) who suggested the term Apoptosis. Significance of apoptosis: Sculpting Developing embryo Maintaining tissue homeostasis
Destruction-Damaged cell (viral infected, DNA damage ,Hypoxia, Nutrient starvation

,Anoikis ,Anoxia)

Abnormality in Apoptosis: Dysfunction or deregulation of apoptotic program brings variety of pathological condition.

Deficient (too little) in apoptosis. Cancer

Autoimmune Eg SLE Systemic lupus erythematosis)

Rheumatoid arthritis
Hoshimoto Thyroidities

Spreading of viral infection

Excessive Apoptosis Neurodegenerative disorder such as Alzhimers disease .,parkinsons disease ,Hungtingtons disease. AIDS Ischemic disease.

Why should cell commit suicide? For proper development : Resorption of tadpole tail at the time of metamorphosis into frog occurs by apoptosis Formation of fingers and toes of the fetus require the removal of tissue between them by apoptosisif it fails syndactyly or polydactyly results. Sloughing of the inner lining of uterus (the endometrium)at the start of menstruation occurs by apoptosis.

 Formation of proper connection between neurons in the brain requires the elimination of surplus cells by apoptosis. Who are encountered through apoptosis? Virus infected cell:Virus infected cell has to be destroyed to prevent sapping further nutrient from the organism or to prevent the spread of viral infection which collapse the normal function of an organ. DNA Damaged cell: to avoid cancer and other genetic abnormalities (on my own) In appropriate immune cell: Durning embryonic development T-lymphocytes are produced that posses receptor capable of binding tightly to proteins present in the surface of normal cell within the body,T-Lymphocytes having dangerous capacity are eliminated by apoptosis What Decides cell to commit suicide:

Withdrawal of positive signal

Continuous survival of most cells requires a continuous signal from other cell.Eg ECM for epithelial cell IL-2 for mitosis of lymphocytes IL-3 growth survival factor for Hematopoietic cell. Receipt of negative signals:

Binding of molecule (death activator) to specific receptor make the cell to commit suicide. Eg: TNF-, lymphotoxin (TNF-), binds to TNF receptor. FAS lignad binds to FAS receptor. Accumulation of unfolded protein, i.e. Protein failed to fold properly into their proper tertiary structure. Damage to DNA, by oxidants, UV, x-Ray, chemotherapeutic drug.

What are the other forms of cell death: Necrosis: It is a form of traumatic cell death,ie cells get injured or cells get punctured ,which will leads to death of a cell by what called necrosis. Paraptosis: cells swell develop bubbles or vacuoles with liquid inside and die this method of suicide called paraptosis. Oncosis: cells expand by taking in lot water in an uncontrolled manner ,soon proteins become denatured, than cells take excess calcium into cells and death follows. Pyroptosis: Pyro relating to fever, ptosis(to sis)-denoting falling,its a pro inflammatory programmed cell death .Macrophages when infected with salmonella or shigella ,caspase 1 gets activated and process the proform cytokines ,IL-1 and IL-18 to their active form which results in pro-inflamatory programmed cell death. Autophagy: An onother form of programmed cell death ,which is characterized by the sequestration of cytoplasam and organelles in double or multimembrane vesicles and delivery to the cells own lysosomes for subsequent degradation by lysosomal Hydrolyses. Autoschizis: Its a bizarre type of cell death. where cell membrane forms cuts or schisms which allow the cytoplasam to leak out,the cell shrinks its size until about only 1/3 its original size and only the nucleus and organelles remain surrounded by tiny ribbon of cytoplasam. If apoptosis is a quiet cell suicide autoschizis is violent, since it slashes itself open violently spilling out its inside.autoschizes shows both apoptotic and necrotic morphological characteristics. Autoschizis happens when cancer cells are treated with Vitamin c & k. Mitotic catastrophe: (Catastrophe sudden disaster )a process involving aberrant mitosis resulting from improper segregation of chromosomes during sister chromatid Generally it is not considered a form of death ,but rather irreversible trigger for death. Senescence: cells fail to divide beyond finate number of population doubling or division, considered a type of cell death in the context of cancer therapy. separation.

How one can come to know that the given cell is alive or death without its division? Interestingly,the shape that cells assume as they attach to the ECM can determine whether they live or die,cells that flatten and spread on the ECM to maximize cells surface attachment survive ,whereas the rounded cells do not ,even though they may remain attached to the ECM. What are the pathways involved in apoptosis Intrinsic pathway Extrinsic pathway Cytotoxic T lymphocytes (caspase independent ) ER-stress mediated pathway INTRINSIC PATHWAY: Intrinsic pathway is also known as mitochondrial mediated pathway,it is activated in response to intracellular stress signals-which include DNA damage High level of reactive oxygen species Viral infection Activation of oncogenes Irreparable genetic damage Lack of survival signal (absence of growthfactor) Severe oxidative stress i.e. The production of large number of destructive free radical. Extremely high concentration of cytosolic ca2+.

Bcl2 family; Activation of the intrinsic pathway is regulated by members of the Bcl2 family of protein,Bcl-2 family members can be sub divided into 2 groups Pro-apoptotic members that promote apoptosis eg (Bad,Bax,Bid ,Bak) Anti apoptotic members that protect cell from apoptosis eg(Bcl-XL,Bcl-w,Bcl-2,Mcl-1& Bf11/A1) Bcl-2 protein contains from 1 to 4 Bcl-2 Homology (BH) domains-Dictates whether the proteins are proapototic or antiapoptotic.Proapoptotic members lack the BH4 domain Antiapoptotic Bcl2 members contain all four BH domains.The sensitivity of cells to apoptotic stimuli can depend on the balance of proapoptotic and antiapoptotic Bcl-2 protein. Where there is excess of proapoptotic proteins (in activity) the cells are more sensitive to apoptosis. Where there is an excess of anti-apoptotic proteins the cells will tend to be more resistant. The proapoptotic Bcl-2 proteins are often found in the cytosol, in an phosphorylated form Eg:Bad is phosphorylated in the presence of IL-3 .The signals from IL-3 receptor causes phosphorylation of Bad.The phosphorylated Bad is sequested in cytosol, encapsulated by an adaptor protein termed 14-3-3, and thus unable to induce apoptosis.Following cellular stress the phosphorylated Bad is de-phosphorylated and they (Bad)are relocated to the antiapoptotic protein disrupts the normal function of the anti-apoptotic Bcl-2 protein. Oligomerization of Bax and Bak in mitochondrial outer membrane to activate surface of Mitochondria,where the anti-apoptotic proteins are located.The interaction between pro and

MOMP(Mitochondrial outer membrane permeabilization) or in the formation of pore in the mitochondrial membrane called the permeability transistion pore or PT pore.Through the pores they permit the release of apoptotic factor such as cytochrome C,apoptosis inducing factor (AIF),SMAC/DIABLO,which resides in the intermembrane space of mitochondria. Once the cytochrome C has been released into the cytosol,it is able to interact with protein called Apaf-1(apoptotic protease activating factor-1)to form multiprotein complex known as apoptosome,this leads to the recruitment of procaspase 9.Caspase belong to the group of enzyme known as cysteine protease ,exist within cell in an inactive proforms or zymogens.Procaspase 9

are thought to become activated into caspase 9 by simply joining the multiprotein complex apoptosome.Caspase 9(initiator of caspase)cleaves and in so doing activate caspase-3 and caspase-7(the effector or executioner caspase).Once effector caspase -3 is activated it leads to apoptotic outcome Apoptotic outcome: Fragmentation of DNA, Nucleus fragmentation, Chromatin condensation, Cell blebbing, Cyotoplasmic convolution Fragmentation of DNA: Fragmentation of DNA into nucleosomal units ie 180 basepair is caused by an enyme known as CAD-caspase activated Dnase. During apoptosis ICAD,is cleaved by caspase such as caspase-3 to release CAD-rapid fragmentation of the nuclear DNA follows. Although cell death can occur without significant DNA degradation why the DNA has to be degraded that to for 180 base pair,because it facilitates the engulfment of resulting apoptotic corpses by phagocytes and eliminates the transforming potential of any damaged or mutated DNA. Albeit several protein participate in cleavage reaction,DNA fragmentation factor DFF is one of the major endonuclease G responsible for intranucleosomal DNA cleavage during apoptosis. In normal or non-apoptotic cell DFF exist in nucleus as a heterodimer, composed of DFF45/ICAD Chaperon and inhibitor subunit and DFF40/ICAD latent nuclease subunit.

Chromatin condensation & nuclear fragmentation: Break down of structural nuclear protein lamins (lamins are intra nuclear protein that mediate shape of nucleus and interactions between chromatin and the nuclear membrane)by caspase 6 results in chromatin condensation and nuclear fragmentation. Cell blebing Activated caspase -3 cause membrane blebbing(Via Rock-1,Rho associated coiled-coil forming kinase-1)cleavage that leaves the kinase constitutively active ,permanently phosphorylating myosin light chain.Blebs is the irregular bulge of plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasmamembrane.

Inactivation of enzyme involved in DNA repair: The enzyme PARP (Poly (ADP-ribose polymerase)is an important DNA repair enzyme and was one of the first enzyme identified as a substrate for caspase.the ability of PARP to repair DNA damage(fragmented DNA to nucleosomal size ) is prevented following cleavage of PARP by caspase-3. Apoptotic bodies engulfment: As cell execute apoptotic programme they lose contact with neighbours and start to shrink. Finally the cell disintegrates into a condensed membrane enclosed apoptotic bodies. This entire apoptotic programe can be executed in less than an hour. Apoptotic bodies are recognized as eat me signal by specialized macrophages, with the aid of phosphatidyl serine on their surface. Phosphatidyl serine is a phospholipid that is normally present in their inner leaflet of plasma membrane. During Apoptosis, a phospholipid scramblase moves phosphatidyl serine molecule to the outer leaflet of plasma membrane. A phosphatidyl serine (PS) receptor is involved in phagocytosis of apoptotic cells. Usually PS is maintained at the inner layer of the plasma membrane (PM) by the action of an ATP-dependent PS-flippase,this flippase is inactivated by caspases, and a scramblase is activated, leading to redistribution of PS to the outer leaflet of plasma membrane. Phosphatidyl serine in not a noxious content. In necrosis lysis content is released. Phagocytosis of apoptotic bodies result in the release of anti-inflamatory cytokines and immune tolerance. Invitro: secondary apoptosis or apoptotic necrosis In the absence of phagocytosis or neighbouring cell apoptotic bodies may proceed to lysis, known as secondary necrosis(secondary apoptosis) or apoptotic necrosis, this process is seen in cultured cells that are undergoing cell death by apoptosis, in vitro. Invivo: secondary apoptosis or apoptotic necrosis Secondary apoptosis may be seen during massive local apoptosis where phagocytosis and neighbouring cells may be unable to cope with the load of apoptotic cell Secondary apoptosis is

detected using Propidium iodine and Annexin V. Neighbouring cell good eg: Ingestion of apoptotic renal tubular cell by adjacent healthy epithelial cell Phagocytes: by macrophages happens in the site where load of apoptotic cell is great at thymus ,lymph node,bone marrow,and at the site of inflammation . Extrinsic Pathway (or) Receptor Mediated pathway: The receptor triggering this pathway are located in the plasma membrane of the cell that is to undergo apoptosis and they are activated by extracellular ligands, Binding of death inducing ligands to cell surface receptors called death receptors. This ligand can be either soluble factor such asTNF,FAS lignad,TRAIL,or can be expressed on the surface of cell such as cytotoxic TLymphocytes Eg:Granzyme ,perforin. Binding of ligand to death receptor can lead to the generation of ceramide typically produced by acid sphingomyelinase, this ceramide release is thought to promote lipid raft fusion which results in a large scale clustering of the death receptor. This large scale clustering of death receptor is important because it helps to amplify the apoptotic signaling. In the absence of amplification of the signaling pathway is needed to activate full apoptotic response. TNF-receptor signaling TNF is produced by T-cells and activated macrophages in response to viral infection or toxic chemical agent such as those used in cancer chemotherapy,elevated temperature,exposure to ionizing radiation. Binding of TNF- to TNFR1,which is present in plasma membrane as pre assembled receptor clustering some cells such as lymphocytes are still able to trigger apoptosis ,but in most cases

trimer.binding produce change in conformation of the receptor death domain. The cytoplasmic domain of each TNF receptor subunit contains a segment of about 70 aminoacids called a death domain, that mediates protein protein interaction. The change in conformation of receptor death domain allows binding of an intracellular adaptor molecule called TRADD (TNF-R-Associated Death Domain (or) TNF-receptor associated death domain)

As TRADD gets binded, interactions happens between death domain and TRADD, This interaction make TRADD to recruit number of different protein. Recruitment of TRAF2 can lead to activation of NF-Kb and JNK pathway. Recruitment of FADD (FAS-associated death domain) by TRADD or with TRADD-results in recruitment of initiator caspase 8 or caspase 10. This assembly of protein (Receptor ,FADD,and procaspase) is termed DISC-death inducing signaling complex. The pro-caspase-8 molecules are brought into close proximity in the DISC,so that they can transactivate one another, i.e. they undergo autoproteolytic cleavage to form active caspase 8. Active caspase -8 then can directly cleave caspase 3 or other executioner caspase, eventually leading to apoptotic outcome. ? INTRINSIC TO EXTRINSIC How and when ? In some cells caspase-8 can cleave the BH-3 only protein Bid, the resulting truncated Bid (t Bid) then moves to the mitochondria and induce cytochrome C release, leading to activation of caspase 9 and caspase3. This cross talk between the Death receptor and mitochondrial pathway can occur in cells when DISC formation and active caspase 8 are insufficient to activate procaspase-3 ,independently of mitochondria. ? Why dont we use TNF, FAS ligand as a curative agent for cancer. Since the TNF, FAS ligand are toxic when administered systemically which has limited their use as anti cancer treatment. Cytotoxic T cell mediated pathway: Cytotoxic T-cell capable of induce cell death (Apoptosis) of infected somatic cells by viruses, and other pathogens (or) Tumor cell. When exposed to infected /dysfunctional somatic cells, cytotoxic T cells release cytotoxins perforin and granulysin (or Granzyme B(GrB).Perforin forms pores(16nm) in the target cells plasma membrane ,allowing granzyme, type of serine protease ,to enter the target cell. Granzyme B can directly activate caspases,which in turn induce apoptosis. Granzyme B can also bypass caspases by directly cleaving ICAD. which in turn induce apoptosis

? what makes cytotoxic Tcell to take granule exocytosis pathway and membrane bound ligand pathway (Or) ?when do cytotoxic T cell release Granzyme and perforin Tumor cell express Non-functional FAS receptor or soluble forms of FAS receptor that will sequester the FAS ligand or expression of FAS ligand on tumor cell. At this condition, exocytosis pathway happen . ? What condition it tooks by pass pathway or direct pathway or what factors guides them to take bypass pathway. Granzyme: A Granzyme A is also important in cytotoxic T-cell induce apoptosis ,activates caspase independent pathway. Once in the cell ,Granzyme A activates DNA nicking Via DNAse NM23H1,a tumor suppressor gene product ,In tumor cell ,Nucleosome assembly protein SET normally inhibits the NM23-H1 gene ,so the cancer cell are immune survivellance. Granzyme A protease cleaves SET complex,thus releasing inhibition of NM23-H1 resulting in apoptotic DNA degradation . SET complex maintain DNA and chromatin structure integrity ,inactivation of this SET complex(SET-NM23H1),contribute to apoptosis by blocking the maintenance of DNA and chromatin structure integrity. Second way: ligand mediated A second way ,when cytotoxic T cell recognize (bind to )their target cell, this binding or intraction activate cytotoxic T cell, activated cytotoxic Tcell starts to express surface protein FAS ligand (FASL),which binds to FAS molecule expressed on target cell (eg:virus infected cell)leading to its death by apoptosis.

ER Stress mediated pathway: ER serves many specialized function in the cell including Calcium storage, lipid synthesis, protein folding into tertiary and quaternary structure of plasma membrane ,golgiapparatus and lysosomes Any factor that disturbs this function of ER is called ER stress. Some biochemical & physiological stimulai such as perturbation(small changes) in calcium Homeostasis Redox status Elevated protein synthesis Expression of misfolded protein Sugar/glucose deprivation Altered glycosylation Overloading of cholesterol.

ER stress is caused by excessive protein traffic-usually caused by viral infection (Or) By accumulation of unfolded protein aggregates, aggregated protein is toxic to cell. To combat this cell posses UPR (unfolded protein response),a pathway to restore ER homeostasis or ER normal function. ER transmembrane receptor detects the onset of ER stress and initiate unfolded protein response. 3 ER transmembrane receptor (or) stress receptors

 Pancreatic ER Kinase ATF-6 IER1 In resting cell the 3 ER stress receptor are maintained in an inactive state through their associated with GRP78,( ER chaperons) On accumulation of unfolded protein or misfolded protein GRP78 dissociates from the 3stress receptor-which leads to their activation and triggers UPR related gene such as chaperon,genes for protein degradation, and p58IPK,genes for aminoacid transport and synthesis. At resting condition: some of the Bcl-2 family members (Bcl-2, Bik, BAX, BAL) are the regulators of mitochondrial induced apoptosis, also reside on the ER. The pro-apoptotic Bax and Bak are kept inactive by interacting with BcL-2 both on mitochondria and ER membrane. BIM is bound to dyenin motor complex of the microtubule cytoskeleton.Calcium(ca2+) primarily stored in the ER by SERCA(sacroplasmic endoplasmic reticulum ca2+ ATpase) that pumps ca2+ into ER. At ER stress condition: Persistant stress triggers the dissociation of GRP78 from IRE1(A encdoplasmic reticulum stress receptor),its dissociation activate IRE1 activated IRE1 recruits TRAF2 (TNF-receptor associated factor2). The IRE1-TRAF2 complex formed during ER stress can recruit ASK1(Apoptosis signal regulating kinase)as a result activation of ASK1 take place,ASK1 activates MKK3&MKK6 that targets P38 MAP kinase and also MKK4and MKK7 that target JNK,it is not known whether p38 kinase has prosurival role, However JNK activation mediate pro-apoptotic effects by phosphorylation of Bcl2,which occurs primarily at the ER,suppress the antiapoptotic activity Bcl-2. Besides Bcl-2,JNK also Phosphorylates BH3(Bcl-2 Homology domain 3) only member of the Bcl-2 family such as BIMwhich enhances the proapoptotic potential. Phosphorylation BH3 is released from Dyenin and translocate to the ER and mitochondria and activate Bax and Bak,which results in cytochrome C release,apoptosome formation & finally apoptotic outcome,whereas Bax & Bak in ER-which is activated by BH3, signalsER to release Ca2+ ,the elevated cytosolic calcium levels leads to activation of calcium regulated protease

called calpains which can inturn activate caspase such as caspase 12 & caspase 4,which can inturn activate effector caspase(3&7) and stimulate apoptosis Caspase 12-In rodent Caspase 4- In human. P53 Master in Blaster: Under normal condition P53 are unstable and present in low level ,presence of P53 protein in the nucleus would inhibit normal cell growth ,cell take care of that by exporting P53 from nucleus to the cytoplasam for degradation by MDM2. When DNA damage occurs through Chemotherapy ,radiotherapy ,UV light ,it activates DNADK (DNA dependent protein kinase )or ATM kinase,which phosphorylates serine 15 within the critical N-terminal region of P53.Which protect P53 from degradation and leaving nucleus as a result protein accumulates within the nucleus. P53 protein posses transcription activation factor ,P53 protein binds to gene to promote transcription of p21,that in turn binds to and inhibit CDK causing hypophosphorylation of RB, thus preventing the release of E2F and blocking G1-S transition ,the p21 gene encodes p21 protein ,which stops cell at G1(growth phase of cell) this allows time to repair DNA just before it enters the DNA replication process. If the repair is not possible,p53 stimulates death promoting gene Bax,,Noxa,PUMA,Bid etc, by binding to its promoter and transcriptionally activate them or inhibit the expression of Bcl-2 antiapoptotic protein. Apoptosis and cancer: Apoptosis escape by virus associated cancer. Human Papilloma virus causes cervical cancer,by producing protein E6 that binds and inactivate apoptosis promoter P53. Epstein barr virus causes Burkits Lymphoma,by producing a protein homology of Bcl-2 Eg BHRF1 protein,this protein can inhibit pro-apoptotic protein such as Bax & Bak-which is essential for apoptosis.

Apoptosis Escape non virus associated cancer B-cell leukemias & lymphomas Express high level of Bcl-2,this high level results from translocation of Bcl-2 gene into enhancer region for antibody production-which avoids apoptosis. Melanoma-inhibit or do not expression of Apaf-1 integral part of apoptosome,(might be due to mutation ) lung and colan cancer secrete elevated levels of decoy molecule(DCR-3) that binds to FASL,plugging it up,so FASL cannot bind to FASr, thus cytotoxic T cell cannot kill cancer cell through the ligand mediated pathway. Some cancer cell express high level of FASL and kill any cytotoxic T cell ,that try to kill them because CTL also express FAS (but are protected form their own FASL) Hepatities B virus -HBx protein,binds to p53 ,and inhibit its transcriptional activity.Cowpox virus-crmA protein inhibits caspase activity. Myxoma virus M-T2 protein binds to TNF thus prevent it from binding the TNF receptor. APOPTOSIS RESISTANCE MECHANISM; IAP Expression of IAP family protein survivin, is highly tumor specific,it is found in most human tumor ,but not in normal adult tissue, and it is expressed high in fetal tissue. Human survivin gene located in chromosome17,it is localized in cytoplasam . IAP(Inhibitor of apoptosis protein)family members-cIAP1,c-IAP2,,cx-IAP(X-chromosome linked IAP) and surviving are potent inhibitor of endogenous caspase ,which results in blockade of TRAIL-induced apoptosis. Specifically IAP, exert their inhibitory activity by interacting with caspase-9,-3,&-7,but not caspase 8 ? Why not to caspase8. .SMAC/DIABLO interacts with IAP family members,antagonizing their inhibitory activity and release the bound IAPs from caspase. Caspase free of IAPs are more susceptible to proteolytic cleavage and activation. The presence of survivin in urine may also act as biological marker for bladder cancer. Surviving confer resistance to anticancer drug eg:Paclitaxel,& Methotrexate.

Xiap X-linked inhibitor of apoptosis protein is over expressed in cells of the H460 cell lines lung cancer.X-IAP binds with procaspase -9 and inhibits its further progression of binding to apoptosome. Resistance mechanism expression of antiapoptotic protein. Some tumors are characterized by the expression of high level of c-flip(c-FLICE like inhibitory protein) Some virus such as herpesvirus,human herpesvirus 8 and moluscum contagious virus also produce DED-containing protein, which are called viral FLICE inhibitory protein(v-FLIP) Elevated level of c-FLIP in tumor tissue may allow tumor cell to escape from the death receptor mediated apoptotic death. c-FLIP,a catalytically inactive homology of caspase-8.FLICE inhibitory protein(c-FlIP) inhibits apoptosis by blocking caspase-8 activation at the death inducing signaling complex.(DISC) .c-FLIP normally competes with caspase-8 for binding to the DISC. Resistance Mechanism Inactivation of proapoptotic genes: Apart from overexpression of proapoptotic gene,tumors can aquire resistance by down regulation or mutation of proapoptotic gene. In certain type of cancer the proapoptotic Bcl-2 family member Bax is mutated,tumor cell lines with mutation of Bax,are more resistance towards apoptosis, Metastatic Melanomas have found another way to escape apoptosis,they do not express Apaf-1 which forms integral part of apoptosome.In Neuroblastoma ,the initiator caspase8 is frequently inactivated by genedeletion or methylation. HSP as apoptosis resistance. ER stress is caused by misfolding of protein which could be repaired by HSP(Heat shock protein),so that the cell death apoptosis is avoided. The HSP -27,70,and 90 play a role in avoidance of apoptosis.HSP prevent the release of cytochrome C and activation of caspases.,Hsp 27,& HSP 70 can inhibit activation of BID by caspase-8 and thereby the extrinsic pathway.

HSP70 & 90 can prevent the assembly/activity of the apoptosome thereby inhibiting the intrinsic pathway, HSP can prevent MOMP by interacting with Bax, Geldana Myc is a therapeutic agent targeted to HSPs and by binding to HSP 90 prevent the interaction with target protein. -------------------------------------------------------------------------------------------------------------------List of Question that arise to rise myself ? ? As the mitochondria is disrupted how do they say apoptosis is active process (Energy involved)? Answer: The mitochondria is not completely destroyed the matrix is not affected, pore forms at the outer layer which does not have its impact in ATP production. ? How does secondary apoptosis or secondary necrosis,apoptotic necrosis is executed all of sudden in cell that is undergoing apoptosis in the absence of phagocytosis or neighbouring cell. ? How does epithelial cell acquires the character of engulfing property of macrophages.and do they recognize apoptotic cell in the same way as if done by phagocytosis, ? Why cant we add or transfer the engulfing property to individual cancer cell within the tumor mass, if we do what would be the outcome ? What factor decides or allow apoptotic cell to choose FADD instead of TRAF2 by TRADD, (Or) ? At what condition TRADD decides to choose TRAF2 , or FADD. ? Why do cytotoxic T cell has to produce two protease, Grb & GrA, among the two different protease, whose participation is more or frequent in making apoptosis on target tumor cell. ? How and why do survivin are more expressed at fetal tissue.

? Why dont survivin expressed from tumor does not act as antigen in adult, Do they induce any immune response once it is expressed from tumor.