The 131 somatic cells die during development of nematode C. elegans revealed a set of genes termed as sed genes cell death which were found in regulating somatic cell death [Ellis and Horvitz.-1986]. Among several two were responsible for cellular death (sed-3 and sed-4) where as sed-9 prevent cell death, by performing antagonistic role [Ellis et al 1991and Hen Gartner et al. 1992]. Sed-4 binds to sed-3 and initiated apoptosis whilst sed-9 binding occur to both protein and inhibit apoptosis [Shaham and Horvitz 1996]. The gene product of Egl-1 does not interfere in most, if not all, somatic cell death (Conradt and Horvitz 1998). Apoptosis is not a passive event but genetically controlled process during the course of evolution, including development, regulation of cellular homeostasis and morphogenesis of multi-cellular organism, immune response to eradicate infected cells. [Krammer 1999, Vaux and Korsmeyer 1999]. However, deregulation of apoptotic pathway contributes to the development of a number of pathological impressions such as autoimmune disease, degenerative disease or cancer [Thompson 1995 Krammer 1999]. The main emphasis was to examine the role of apoptosis in prevention of cancer and their cell lines (Proliferation and development) avoidance of apoptosis is one of six hallmark of cancer. The six hallmark of cancer include-self, sufficient growth signal sustained angiogenesis, tissue invasion and metastasis (Hanhan and Weingerb 2000), accumulation of mutation and further cancer cell promotes more mutations to avoid "cell suicide" (Evan and Littlewood 1998). Thus exploration of apoptotic pathway will enlighten and improve the treatment methods targeting the process in either positive or negative fasion.
Caspases: the executioners
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Executioner's caspases family highly conserved cysteine proteases called as caspases for cysteine aspartate- specific proteases, deal a broad range of execution phase of apoptosis (Alnemri et al. 1996, Salvesen and Dixit 1997, Thornberry and Lazebnik 1998). The caspases were recognized from the observations of common stepwise manner when a Cell / Tissue /organ exposed to variety of death stimuli (Kerr et al. 1972). Presently, the number of mammalian caspases are 14 but 13 actually because of caspase-13 was miss identified and it was bovine caspase (Koenig et al. 2001), including 10 in human's interleukin 1Beta- converting enzyme (ICE) was known first mammalian caspase and named as Caspase-1 which generates inflammatory response (Thornberry et al 1992). Caspase-1 has sequence homology with C. elegans cysteine proteases sed-3 and first time implicated in apoptosis (Yuan et al 1993). All multi-cellular organisms were exhibit almost similar caspase homologies that have been examined. They were known as hallmark of apoptosis that was also came from following observations: (i) Low expression of hsp70 (ii) Low expression of anti-apoptotic factors (iii) High expression of pro-apoptotic factors (iv) High expression of tumor suppressor p53 (v) Activation of executioner caspases.
Several studies upon knockout mice suggested in vivo role of some caspases during apoptosis (Zheng, T.S. and R.A. Flavell 2000). In a report, caspase-3 and caspase-9 null mice exhibit severe defect during developmental processes (Kuida et al. 1996, 1998), whilst caspase-8 null mice exhibit severe apoptotic phenotype and die in utero. Although highly expressed caspases lead to apoptosis, in vitro studies on caspase-1, caspase-11 and caspase-12 knockout mice suggested normal development, mean these caspases are not necessary for apoptosis, at least during developmental process [Kuida et al. 1995, Wang et al. 1998b]. On the basis of their function which they perform, mammalian caspases can be categorizes into two. Former one promotes activation of proinflammatory cytokine. Secondly caspases-1, caspases-4, caspases-11, caspases-12 with initiator caspases (caspases-8, caspases-9, caspases-10) and effectors (caspase-3, caspases-6, caspases-9), caspase-14 associated with apoptosis in the skin. Caspases are inactive zymogens (~30-50kDa) having three variable domains, N-terminal pro-domain a large (20kDa) cysteine on active site, a C-terminal (10kDa) small domain [Thornberry, N.A. and Y. Lazebnik 1998, Wolf and Green 1999] and a highly variable N-terminal domain involved in the activation of caspases. Effector caspases have small N-terminal domain in contrast to initiator (apical) caspases and some time a caspase recruitment domain (CARD), executioner caspases (DED) and caspases-8, caspases-10 involved in protein-protein interaction. Autolytic cleavage of caspases induce their processing which transmit downstream signals of cell death (Slee et al. 1999a, Slee et al. 1999b). Evidences from several in vitro studies suggested two model one is apoptotic signal pass through adaptor protein to initiate apical caspase ultimately cell death. Further positive feedback loop created through autolytical cleavage of apical caspases through activated executioner caspases such as caspases-3, caspases-6 and caspases-7. The proposed models were also supported by several studies on knockout mice (Kuida, K., T.F. Haydar 1998).
The Intrinsic Apoptotic Pathways
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Pathophysiological and environmental stressors induce activation of intrinsic apoptotic pathway. The intracellular signaling mechanism of apoptosis is remaining to be investigated. However, all of these stressors in mammalian system or in vertebrates aim to change mitochondrial membrane potential (Ïˆâˆ†m) and/or release of cyt-c from inter membrane space (IMS) into cytoplasm as a result activation of effector caspases cascade, cell death ultimately. Evidence from several new studies on involvement of mitochondria in apoptosis shown by cell free system, where cell death needed the presence of mitochondria, release of cyt-c and processing of caspases (Newmeyer et. al. 1994, Liu X., C. Naekyung Kim 1996). Further evidences suggest that stresses beyond threshold limit can induce release of cyt-c from mitochondria (Kluck et al. 1997, Yang et al 1997). Indeed cyt-c, a number of other proteins with pro-apoptotic nature are also associated with release of cyt-c from mitochondria during apoptotic process (review - Wang). In addition, apoptosis is a result of dysfunction in mitochondrial metabolism includes electron transport dysfunction, oxidative phosphorylation and ATP metabolism, change in redox potential (Green and Reed et al. 1998). However the question remain unclear which of these mitochondrial events are a consequence and which a cause of apoptosis is? The Bcl2 family is well known for regulation of mitochondrial events which transmit both cell damage and cell survival signal. Anti-apoptotic factors of Bcl2 family maintain mitochondrial membrane permeability (MMP) whereas pro-apoptotic factors induce formation of transition pore in mitochondrial membrane and thus apoptosis.
The Bcl-2 family of proteins
B cell Lymphoma 2 (Bcl-2), a proto-oncogene was first discovered by its involvement in human follicular lymphoma where it found attached to immunoglobulin locus by chromosome translocation (Tsujimoto 1984, 1985). Bcl-2 has potential to prevent apoptosis in response to a number of physiological and pathological stimuli, thus connecting inhibition of apoptosis with tumor progression (Vaux et al 1988). Bcl-2 protein and Ced 9 of C. elegans found very much similar to each other during a development of C. elegans. Apoptosis is prevented via Bcl-2 protein family providing further evidence that the mechanism is highly conserved during the course of evolution. In mammalian system there are about 20 member identified, each posses at least one of four conserved Bcl2 homology domains (BH1-BH4) (review-Adams and Cory 1998, Cory and Adams '2002). Bcl2 family member can be classified on the basis of their structure and function such as pro-survival member having BH1 and BH2 domain and pro-apoptotic members Bax, Bok, Bak which share homology in BH1, BH2 and BH3 domains but not in BH4 indeed some having BH3 only includes Bid, Bim, Bad and Noxa. The role of BH3 domain is thought apoptotic (Chittenden et al. 1995) but both type of factors necessary for apoptosis (Zong et al 2001). However, Bcl2 family members having C-terminal hydrophobic transmembrane domain and N-terminal domain organization, C-terminal domain involved in the formation of integral proteins on cytoplasmic faces of intracellular membranes such as mitochondria, ER, and nuclear membrane (Cory and Adams 2002). Further, in the absence of apoptotic stimuli the pro-apoptotic members attached to cytosolic surface and with cytoskeleton components (Hsu-1997 Gross- 1998 Puthalakath 1999).
Stressors such as a number of cytotoxic insults, UV radiation, Cytokine withdrawal and cytotoxic drugs are strongly inhibited by Bcl-2 and its closest relative Bcl-xL and Bcl-w (Cory and Adams 2002). Anti-apoptotic factors of Bcl2 family interact with outer mitochondrial membrane and prevent release of cyt-c from inner mitochondrial membrane into the cytoplasm by maintaining mitochondrial membrane-integrity, and thus inhibit apoptosis (Krajewski et al. 1993 de Jong et al. 1994, Susin et al. 1996, Kluck et al. 1997 Yang et al. 1997). Heterodimerization of Bcl-2 and Bcl-xL combiningly prevent loss of mitochondrial membrane potential by inhibiting membrane permeability transition (Shimizu et al. 1996, Susin et al. 1996, Zamzami et al. 1996). Structural studies of Bcl-xL and Bcl-2 enlighten the mechanism of anti-apoptotic factor are thought to use predominantly to inhibit apoptosis (Muchmore et al. 1996, Sattler et al. 1997, Petros et al. 2001). Hydrophobic binding pocket for BH3-only family members was created by BH1, BH2 and BH3 domain residues. Therefore pro-apoptotic and anti-apoptotic factors physically interact to form heterodimers and the ratio of anti-apoptotic to pro-apoptotic factors function as an equilibrium state that sets threshold of susceptibility to apoptosis through intrinsic pathway (Oltvai et al. 1993, Oltvai and Korsmeyer 1994). Integration of downstream signaling of apoptosis leads to post-translational modulations that alter the conformation and affinity of binding between proteins, permitting the release of apoptogenic factor and activation of pro-apoptotic factors.
BH3 only family
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In intrinsic pathway of apoptosis BH3-only family members exhibit upstream to integrate signals in apoptosis machinery (Huang and Strasser 2000; Kroemer and Reed 2000; Scorrano, L. and S.J. Korsmeyer. 2003). Bim acts as a sensor of cytoskeleton integrity (Puthalakath et al), Bid responsible for death receptor signaling (Li et al 1998, Luo et al. 1998b), and Bad acts as a sensor for growth factor withdrawal (Soane et al 2001). The broad range of BH3-only proteins involved to allow monitoring different sub-cellular compartments and acts against several stressors (Huang and Strasser 2000). Introduction of death signal activate by BH3-only protein via transcriptional regulation and post translational modification of BH3-only members. Puma, Noxa and p53 targeted genes up-regulated transcriptionally through BH3-only family (Oda et al. 2000; Nakano and Vousden 2001), whereas post translational modulations includes inactivation and proteolytic cleavage, may induce formation of active conformations that alter sub-cellular localization and promote binding to other proteins (Zha et al. 1996, Li et al. 1998, Luo et al. 1998 ,Gross et al. 1999). Activation promotes translocation to the mitochondria where BH3-only proteins exert their biological function. BH3-only proteins are bifunctional, first they interact with hydrophobic groove of anti-apoptotic factors (Bcl2, Bcl-xL) and block their over expression, secondly, activate multi-domain pro-apoptotic factors Bax, Bak and Bcl-xS. Evidences also suggested that some BH3-only family member (OFM) may directly permeabilize the OMM themselves (Grinberg et al 2002). Synthetic BH3 in a model directly activate Bax and Bak whereas other member binds Bcl2 and displace BH3-OFM and neutralizes Bcl2/Bcl-xL by increasing the ratio of Bax/Bak. Recent evidence suggest that Bcl-2, Bcl-xL exhibits antagonistic potential against Bax/Bcl-Xs/Bak (Cheng et al 2001).
Mitochondrial membrane permeabilization by Bax/Bcl-Xs/Bak from previously reported studies indicates that major player between the sensor of intracellular damage are multi-domain pro-apoptotic Bcl-2 member such as Bax, Bak, Bcl-Xs, and BH3-only proteins and mitochondrial membrane permeabilization resulting abrupt ATP metabolism, dysfunction of electron transport system and release of cyt-c from mitochondria (Eskes et al. 1998, Jurgensmeier et al. 1998). Bax/Bak doubly deficient cells genetic studies provide strong evidence that these factors are important although mutually redundant, for mediating release of cyt-c by BH3-only proteins in response to a diverse range of intracellular stresses ( Lindsten et al. 2000,Chang et al. 2001,.Wei et al. 2001, Zong et al. 2001). Although exact mechanism is need to be investigated, but Bax activation leads conformational change and translocation from cytosol to mitochondria where it embedded into OMM (Hsu et al. 1997, Wolter et al. 1997, Gross et al. 1998, Desagher et al. 1999) in the same way bak activation also required conformational change, but it already resident in OMM (Griffith et al 1999). In addition, recent report has been suggest that Bak oligomerization in OMM is depend on change in Bax conformation during activation of it (Mikhailov et al. 2003). Bax/Bak activation require formation of homo-oligomer in the OMM resulting permeabilization of OMM and release of apoptogenic factor (cyt-c) from IMS (Antonsson et al. 2000, Eskes et al 2000; Korsmeyer et al. 2000, Wei et al. 2000).
Cyt-c is a nuclear transcriptional product, transported to mitochondria and bind with heme group to form holo-cyt-c, is an active form. Active form of cyt-c is responsible for initiation of apoptotic events (Yang et. al. 1997). The molecular mechanism of cyt-c release from mitochondria in Bax/Bak oligomerization dependent manner is still doubtful although three models are present today. According to first one Bax/Bak balance the mitochondrial permeability transition (MPT) via interaction with permeability transition pore (PTP) component including voltage dependent anion channel (VDAC) or the adrenine nucleotide translocase (ANT) (Marzo et al. 1998, Shimizu et al. 1999). According to the second model; Bax/Bak oligomers induce formation of lipid channels and affect the permeability of outer mitochondrial membrane (OMM) (Hardwick and Polster 2002, Kuwana et al. 2002). According to third model; Bax/Bak oligomers create de novo pores in the (OMM) (Korsmeyer et al 2000; Wei et al 2001). The peculiar characteristic of MPT induces loss of (âˆ†Î¨m) mitochondrial potential, swelling of mitochondria, uncoupling of oxidative phosphorylation as a result negligible amount of ATP production (Green and Reed 1998) whereas some report show that both pro-apoptotic and anti-apoptotic factor of Bcl-2 family member interact with PTP complex (Tsujimoto and Shimizu 2000 review, Zamzami and Kroemer 2001), other evidence suggest that there was no such interaction observed during this process (Reviewed in Martinou and Green 2001, Mikhailov et al. 2001). Therefore, MPT involvement in the release of cyt-c is doubtful and controversial, with evidence for both (Marzo et al. 1998, Narita et al. 1998 and Tajani et al. 2002) and in contrast (Kluck et al 1997, Bossy-Wetzel et al. 1998 and Skep et al. 1998, Jurgensmar et al. 1998, Wang et al. 1998a) the opening of permeability transition pore complex and loss of âˆ†Î¨m induce the release of cyt-c. Activation of cysteine aspartate protease may involve in the formation of PTP that is blocked with the caspases inhibitor Z-VAD-fmk (Susin et al. 1997). Studies suggests that PTP formation may be the output of a positive feed-forward look rather than a mechanism for cyt-c release that take place upstream caspase activation (Bossy-Wetzel et al. 1998).
The idea of Bcl-2 family members balancing (MMP) mitochondrial membrane permeability through the formation of pores is based on the similarities in the structure of Bcl-xL and diphtheria toxin, which create pore in biological membrane (Kagan et al. 1981, Muchmore et al. 1996). Bcl-xS/xL (Minn et al. 1997) and Bax (Antonsson et al. 1997, Schlesinger et al. 1997) has been found to create pores in synthetic membrane, although physiological presence of Bcl-xL channel is questionable because of low pH activation. Evidence from recent studies suggest that Bax create pore by interacting with lipid and alter membrane curvature but it require co-operation of Bid protein (Kuwana et al 2002, Basanez 1999, 2002). Therefore, molecular mechanism of cyt-c release during apoptosis is a quick, multistep and complete process that takes place some hours before exposure of PS residue and loss of membrane integrity (Goldstein et al.) regardless of nature of initial apoptotic stimuli, suggesting that a common mechanism exists to control release of cyt-c.
Release of cyt-c induces caspase processing and formation of multimeric complex known as apoptosome (Zou et al. 1999). The crucial protein which involved in the formation of apoptosome is cytosolic apoptotic protease-activation factor-1 (Apaf-1). Apaf-1 has similar homology as in ced-4, ced-4 responsible for the death of nematodes C.elegans (Zou et al. 1997). Released cyt-c from inner mitochondrial membrane stimulates complex formation by binding with Apaf-1 resulting oligomerization (Li et al. 1997, Zou et al. 1997, Chcain et al. 2000). The complex induces cleavage of inactive initiator caspases into active initiator caspases such as pro-caspases-9 to caspases-9 by homotypic interaction between CARD domains on N-terminal of Apaf-1 and pro-caspase-9. Activation of caspase-9 is dependent on dATP or ATP hydrolysis, suggesting energy dependent process (Li et al. 1997, Hu et al Saleh et al. 1999, Zou et al. 1999). Caspase-9 activation induces oligomerization at the apoptosome and remains with Apaf-1, function as an allosteric regulator (Srinivasula et al. 1998, Stennicke et al. 1999). Formation of apoptosome and activation of caspase-9 recruited to executioner caspases like caspase-3, caspases-6 and caspase-7.
Apaf-1 consist of two domain organization, one is N-terminal CARD domain (a Ced-4 domain) and C-terminal domain comprise mostly of WD-40 repeats (Zou et al. 1997). The WD-40 repeats domain necessary for protein-protein interaction includes self-association to form the apoptosome and binding to cyt-c (Srinivasula et al. 1998). Two-WD-40 domain covers the CARD domain in an inactive Apaf-1 but is dislodged upon binding of cyt-c to permit the binding of caspase-9. ATP/dATP binding induces conformational change resulting formation of hepatamer in the shape of a wheel, the apoptosome complex (Acehan et al. 2002). Evidences from knockout mice missing the Apaf-1 gene suggest the important role of these proteins in apoptosis (Cecconi et al. Yoshida et al. 1998). Phenotype of Apaf-1 lacking mice similar to that of caspase-9 and caspase-3 null mice, Apaf-1 null mice exhibit excessive number of neurons, facial abnormalities, suppress interdigital webbing and die at embryos or soon after birth. In addition, exposure of UV and gamma-radiation showed defective/abnormal apoptosis. Evidence from recent studies on cyt-c knockout mice, caspase-9, caspase-3 and Apaf-1 knockout mice have been suggested the important role of these factors in the intrinsic apoptotic pathway (Kuida et al. 1996, Kuida et al. 1998). In contrast recent report suggests that apoptosome is not compulsory for haematopoietic homeostasis and is not required for initiation of apoptosis, amplifier of caspase cascade (Marsden et al 2002). The cells Apaf-1-/- undergo apoptosis, albeit in a slow manner, which suggest that the apoptosome merely accelerate apoptosis that is occurring through another ways. Findings from the present studies and other indicate that other caspase like caspase-2 may act upstream of the mitochondria to induce apoptosis in response to intracellular stressors and Bcl-2 might function at this stage to inhibit apoptosis. (Lacana and D A damio 1999, Lassus et al. 2002, Marsden et al. 2002). Caspases activation in C.elegans regulates in similar fashion to that of formation of apoptosome and its regulation through Bcl-2 family members. Ced-9 binding to Ced-4, inactivates it, the homologue of Apaf-1, Egl-1(homologue of BH3-only) induce an apoptosis through binding with Ced-9 and displacing Ced-4, which then migrate to perinuclear region (Contadt and Horvitz 1998). Ced-4 oligomerise and bind to Ced-3 which is inactivated by induced proximity mechanism similar to caspase-9 activation at the apoptosome. Bcl-2 family member Bcl-xL found to associate with caspase-9 and Apaf-1 to inhibit caspase-9 in a ternary complex that structurally and functionally similar to Ced-9/Ced-4/Ced-3 complex present in C. elegans (Hu et al. 1998, Pan et al. 1998b). However, the direct interaction between several Bcl-2 family member and Apaf-1 has not been established yet (Moriishi et al. 1999, Conus et al. 2000, Hausmann et al. 2000). Recent studies suggested that a novel anti-apoptotic Bcl-2 family member bind to Apaf-1 and inhibit caspase-9 activation (Schmitt et al. 2004). Therefore, confirmation of the finding is needed in the view of there significane. In contrast, Ced-4 and Apaf-1 is not located at mitochondria while Ced-4 unlike Apaf-1 does not contain WD-40 domain and thus not bind to cyt-c. Further, need for cyt-c in the activation of caspase in response to stressors appear to be a relatively new addition acquire through evolution.
Other pro-apoptotic proteins
Cyt-c released from inner mitochodrial membrane during apoptosis, induces parallel discharge of many other pro-apoptotic proteins in a co-ordination for example second mitochondrial activator of caspase (Smac), also called as DIBALO (Direct IAP binding protein with low PI) (Du et. al. 2000, Verhagen 2000) Smac/DIABLO antagonising the action of IAPs and form homodimer to induce apotosis. Binding to IAPs, C-IAP-2, C-IAP-2 and survin modulates caspases, either directly competing for binding or indirectly (Du et al. 2000, Verhagen et al. 2000, Wu et al. 2000 and Srinivasula et al. 2001, Silke et al. 2002). Thus, Smas/DIABLO has pro-apoptotic activity which induce apoptosis by enhancing caspase-activation. For This reason DIABLO knockout mice show apoptotic phenotype (Okada et. al. 2002). During apoptosis a 57kDa protein, AIF release from IMS of mitochondria (Susin et. al. 1999, Daugas et. al. 2000). AIF can alter mitochondrial membrane potential (âˆ†Ïˆm) and thus cause release of cyt-c from mitochondria. Further, DNA fragmentation and chromatin condensation were detected by AIF induces cell death (Susin et al 1999, Daugas et al 2000). The fuction of AIF not prevented by caspase inhibitors such as Z-VAD-fink, indicating the mode of cell death is a caspase-independent that show apoptotic feature inducing cell shrinkage, exposure PS and DNA fragmentation etc. (Donovan and Cotter 2004). The AIF in mammalian system and WAH-1 in C. elegans associated with apoptosis (Joza et al. 2001, Wang et al. 2002). In C.elegans Egl-1 promote the release of WAH-1 from mitochondria, which show similar effect like Bid which can promote release of AIF from isolated mitochondria (Van Loo et al. 2002a, Van Loo 2002b).
Endonuclease G (endo G) and Omi/Htr A2 are the protein which release from IMS of mitochondria. The release of couple proteins cyt-c and AIF controlled by Bcl-2 family members. In response to apoptotic signal Endo G release into cytoplasm which is located in IMS of mitochondria and alter mitochondrial proteins, (Li et al. 2001) migrate to nucleus and generate oligonucleosomal DNA fragmentation in the absence of caspase activity or CAD (Van loo et al. 2001). Similar mechanism of DNA cleaveage has been observed in C.elegans homologue, CPS-6 was first mitochondrial protein play crucial role in apoptosis (Parrish et al. 2001). The function of CPS-6 appears synergistically with WAH-1 in DNA fragmentation (Wang et al. 2002).
Serine protease OMI/HtrA2 play pro-apoptotic role through caspase-dependent or caspase-independent mechanism of cell death in mammalian system (Suzuki et al. 2001a, Hegde et al. 2002, Van Loo et al. 2002). OMI/HtrA2 perform dual function of promoting apoptosis by serine protease for antagonism IAP activity in a similar way to Smac/DIABLO (Suzuki et al. 2001a, Hegde et al. 2002, Verhagen et al. 2002). Therefore, mitochondria is a center of intrinsic apoptotic pathway. Fig.1,2,3. Dysregulation of mitochondrial functions result into loss of mitochondrial membrane potential, release of cyt-c and a number of other apoptogenic factor into cytosol which cause cell death either by caspase-dependent or caspase-independent means. In comparision with the role of cyt-c in apoptosis, these factor may represent more ancient machinery used in programmed cell death but mechanism of action and their release are not well understood. However, it is clear that Bcl-2 family members regulate apoptotic pathway(s).
The extrinsic or death receptor pathway
Cell death receptor pathway is an instructive death mechanism that is essential in the immune system (Ashkenazi and Dixit 1998). Cell death through extrinsic apoptotic pathway takes place by the activation death receptor such as Fas/Apo-1/CD95; TNF-R1 (Tumor necrosis factor receptor-1), DR3 (Death receptor -3), TRAIL-R1 (TNF-related apoptosis inducing ligand receptor-1) also known as DR4 (Death receptor -4), TRAIL-R2 (DR5), DR6, T75-NGFR, TNF/NGF-receptor super family (Reviewed in Wajant 2003). Apoptosis induced by death receptor pathway seems to be more recently evolved and likely to be alongside the development of the immune system is more complex organisms. The unique characteristics of death receptor are a conserved cytoplasmic death domain, which is induces downstream execution of apoptosis events (Feinstein et al. 1995, Nagata1997). Receptor mediated signaling is most frequently achieved through the binding of corresponding ligands which form a part of the TNF-family ligands. Initiator caspases in extrinsic apoptotic pathway get activated in a complex before the proteolytic cleavage and downstream activation of effectors' caspases in a similar fashion such as in intrinsic apoptotic pathway. The extrinsic pathway is not affected by Bcl2 family members, however cross talk can occur between extrinsic and intrinsic apoptotic pathways through BH3-only protein Bid (Roy and Nicolson 2000). Each of these aspects will be discussed in more detail below with a particular reference on death receptors Fas/TRAIL-R1 TRAIL-2 and their corresponding ligands.
The death receptor/Ligand system
The super family member of TNF-R are known as a type of membrane proteins characterized by the presence of six cysteine residue rich repeat in their extra cellular domain, which participate in ligand binding (Smith et al. 1994). Further, as mentioned the subset that involved in the formation of death receptor, consist an intracellular death domain (DD) (Feinstein et. al. 1995). It is one of 4 death fold motifs, which include CARD, DEP and PYRIN (Reviewed in Lahm et al. 2003). These domains involved in homotypic protein-protein interactions, such as CARD-CARD has been shown interaction between Apaf-1 and caspases-9. DD present in those protein which are found in vertebrates predominantly and essential for both apoptosis and NF-kB signaling in mammals but only latter in Drosophila (Lahm et al 2003). Almost all ligands for death receptor signaling evolved from TNF-family, expressed as type-2 membrane-protein and become organized as homotrimers (Smith et. al. 1994, Bodmer 2002). Proteolytic cleavage through metalloproteases or by alternative splicing produces a soluble form of ligands in many cases. It was reported that a soluble form of ligand is not sufficient to induce signaling but able to activate the receptor with comparable efficiency to the membrane bound in some cases, (Wajant 2003). Homotrimer ligand bind to three molecules of its corresponding receptor, which are pre-assembled in signaling incompetent complexes via an N-terminal domain called the PLAD (Pre-ligand binding assembled domain) (Papoff et al. 1999, siegel et al. 2000). The PLAD has cysteine rich repeat, not overlap with extracellular ligand binding region and responsible for receptor signaling. Conformation change in the PLAD assembly complex induces formation of death inducing signaling complex (DISC) at the cytoplasmic death domains of the receptor (Kischkel et al. 2001).
The DISC vs apoptosome of extrinsic pathways
We know more about DISC from the signaling studies by Fas, although it just like as DR3, TNF-R1, TRAIL-R1 and TRAIL-R2. Formation of DISC need at least a ligand-receptor trimer (Kichel et al. 1995b) but there is also evidences of trimer ligand receptor complexes aggregating into supramolecular clusters (siegel et al. 2000). Ligation of a pre-associate death receptor stimulates probability to recruit this cytoplasmic adopter protein Fas associated with death domain (FADD, also known as MORTI) (Boldin et al. 1995, Chinnaiyan et al. 1995, Kischkel et al. 1995b). FADD molecules has C-terminal domain that form homophilic association with DD on cytoplasmic tail of ligand receptor (Chinnaiyan et al. 1905). Whilst N-terminal of FADD consist a second death fold motif, a DED which help in binding of caspases monomer to N-terminal DED domains in another homophilic interaction, resulting a localized interaction of caspases-8 (Boldin et al 1996, Muzio et al. in 1996).
Integration of downstream signaling form DISC stimulates caspases-8 activation, most probably dimerization (Muzio et al. in 1998). Autolytic cleavage of caspases-8 leads its activation and heterotetramerization in cytosol upon signal from DISC (Medema et al. 1997). Many reports suggested that active tetramer is formed and still attached to the DISC (Lavrik et al 2003). Evidence from recent reports suggested that cleavage is neither sufficient nor necessary for catalytic activity and serves only to stabilize caspases-8 dimer at DISC (Boatright et al. 2003). According to current thought active caspases-8 dimer may be retained at the DISC, whilst fully cleaved tetramer is released. In conclusion, different form caspases-8 has different target substrates (Thorburn 2004). Regardless caspases-8 activation, initiates caspase processing by inducing downstream effector caspase in a similar fashion that of caspses-9 (Enari et al 1996). In group of initiative caspases, caspases-8 studied and contains DD domains other being caspases-10, seem to be the main initiative caspases recruited the DISC in Fas signaling (Peter and Krammer 2003). Evidences from recent reports suggest that caspases-10 recruited and activated TRAIL-R1, TRAIL-R2 and Fas, DISC although it cannot functionally substitute (Kischkel et al 2001). DISC becomes activated through the association of FADD and caspses-8/10 in a complex with DD of a ligated death receptor trimer. Therefore, DISC is a initiating complex of extrinsic apoptotic pathway similar to Apaf-1/cyt-c/Caspase-9 apoptosome in intrinsic pathway.
Cross talk with intrinsic pathways
Activation of extrinsic apoptotic pathway can leads to the recruitment of intrinsic apoptotic pathway through caspases mediated cleavage of BH3-only proteins, expression/down-regulation of Bcl2 family member, and Bid activation (Li et al. 1998, Luo et al. 1998b). Cleaved form of Bid (t-Bid) migrate to the mitochondria and activate Bax and Bak, Bcl-Xs these induce mitochondrial membrane permeabilization and release of apoptogenic factors such as cyt-c (Desagher et al. 1999, Eskes, et al. 2000, Wei et al. 2001). In contrast activation of intrinsic apoptotic pathway Bid was found sufficient to induce apoptosis of target cell (Scaffidi et al. 1998). Apoptosis of these cell can be prevented by over expression of Bcl2 or Bcl-xL due to inhibition of mitochondrial events. In contrast type-2 cells do not require intrinsic apoptotic pathway to undergo apoptosis and apoptosis cannot be inhibit by down-regulating the expression of Bcl2 and Bcl-xL, release of apoptogenic protein and discrete activation of caspase-8 at the DISC induce recruitment of intrinsic apoptotic pathway (Scaffidi et. al. 1998). Formation of DISC and activation of caspases-8 in type-1 cells lead to direct cleavage of caspases-3 and activate cell death mechanism. In addition, DISC formation has been found lower in type-2 cells and a small amount of caspases-8 is produced at DISC which is not sufficient to induce apoptosis through activation of executioner caspases. Evidences from studies in gleoma cell line support the model of differential activation of caspases-8 in type 1 and 2 cells (Knight et al. 2004). However, studies also suggest that the level of XLAP may determine whether a cell require recruitment of intrinsic apoptotic pathway or not (Bratton et al 2002, Bratton and Cohen 2003). Conclusion from the above studies suggested that type-2 cells had high levels of XLAP and required release of Smac/DIABLO from mitochondria whereas type-1 cell show low level of XLAP which does not needed Smac/DIABLO release from mitochondria. Although, activation of apoptotic pathway recapitulated to each other upon the trimerization of Fas-L (Schmitz et al. 1999) others have shown when multimerised Fas-ligand is used to stimulate apoptotic pathway, Bcl2 and Bcl-xL fail to inhibit apoptosis (Huang et al. 1999, 2000). Evidences from Huang and Colleagues suggest that membrane bound Fas-L is use to stimulate cell death pathway trimerised ligand do not.
In vivo studies with knockout or transgenic mice provided evidence to the model. Thymocyte from Bid deficient, Bax/Bak doubly deficient or Bcl2 transgenic mice remain sensitive to Fas-mediated apoptosis (Recapitulating the type 1 phenotype). Whilst hepaitocytes resistance to Fas-signaling and thus are type-2 cells (Strasser et al. 1995, Lacronique et al 1996, Rodriguez et al. 1996, Yin et al. 1999, Lindsten et al. 2000, Rodenques et al. 1996, Wei et al. 2001). But recent report suggested the Bcl-2 transgenic mice exhibit sensitivity to Fas mediated apoptosis because receptor is engaged with oligomerized Fas ligand (Loo et al. 2003). Loo and Colleages reported that in vivo treatment of anti-Fas antibody induces apoptosis of hepatocyte which can be blocked by Bcl2 (i.e. a type- 2 pathway), whilst accumulation Fas ligand induces apoptosis that cannot be affected by Bcl2 (i.e. a type 1pathway).
Comparing with intrinsic apoptotic pathway, receptor mediated apoptosis seems to be more recently evolved form of activating cell death pathway in higher organisms. Death receptor involved in apoptotic pathway of immune system and in immune response co-related extrinsic apoptotic pathway and immune system evolved in concert with each other. However, the two pathways are not entirely exclusive to each other as mentioned by the ability of caspase-8 to active pro-apoptotic Bcl2 family member Bid which connect it to mitochondria mediated intrinsic apoptotic pathway.
Non-classical apoptotic cell death
Necrotic cell death is energy independent and misregulated type of cell death, characterized as swelling of cellular organelles and cytoplasm which leads to release of cellular content in to surrounding tissues, as a result inflammatory response is generated. Necrosis cell death is just opposite to apoptotic cell death. Necrosis is also caused when tissue /cells got severe injuries that may be pathological conditions (Galluzzi et al. 2007). Necrosis can also be programmed in certain condition that was revealed by several recent studies. Therefore, necrosis is the result of imbalance signaling from normal apoptotic signaling. The signaling mechanism involved in necrosis is structurally and functionally need to be investigated but some important work have been made such as RIP kinase has been found associated to Fas induces necrosis. Necrotic cell death was accelerated by production of calcium and ROS as a result execution of necrosis.
Necro-apoptosis is a form of physiological cell death characterized by the execution of both necrosis and apoptosis simultaneously. The term necro-apoptosis enlighten a common pathway resulting occurrence of both form of cell death (Lemasters, 1999; Lemasters et al, 1999; Jaeschke and Lemasters, 2003; Kim et al, 2003). Necro-apoptosis associated with apoptosis, during loss of mitochondrial membrane potential, formation of transition pore in mitochondrial membrane resulting depolarization and uncoupling of oxidative phosphorylation. Further, uncoupling of oxidative phosphorylation leads loss of mitochondrial membrane potential, dysregulation of electron transport system, release of cyt-c due to unavailability of ATP. Therefore, execution of apoptosis and necrosis is directly depending upon the availability/unavailability of ATP in mitochondria/cytosol because increased level of ATP in cytosol inhibits release of cyt-c and formation of mitochondrial transition pore. Hence, the process is accelerated by increased level of ROIs, Ca++, oxidation of pyridine nucleotide and glutathione in mitochondria. Although, ATP level is the determining factor whether a cell undergo apoptosis or necrosis (Nicotera et al, 1998; Eguchi et al, 1999). When cells adopted apoptotic pathways it expresses phagocytic markers on their surface and phagocytosed by scavengers cells as a results no inflammation does occur whereas inhibition of effector caspase resulted diversion from normal apoptotic pathway. Therefore, some authors termed it as apo-necrosis (Papucci et al 2004).
An evolutionary and genetically fixed process which helps to eliminates damaged or nondysfunction cell component and maintain cellular homeostasis. Autophagy is necessary for differentiated development and survival of host and slow process but shows adaptive response against several pathological conditions such as cancer infection and degeneration of neurons. Autophagy can be classified as macroautophagy, micro autophagy and chaperon modulated autophagy (CMA). Large structure were degraded by macro-micro autophagy but phagosome is the double membranous structure and responsible for degrading cellular protein/organelles. In contrast micro-autophagy and CMA associated with incorporation of substrate and target-proteins into lysosome (Mizushima et al 2008). Lack of nutrient and growth factor deprivation autophagy sustain cell survival by degrading waste cellular components whereas uncontrolled up regulation of autophagy results may be cell death and these over expressed ATGs eg. Becline-1 leads death of mammalian cells (Pattingre et al 2005). Danger signal from autophagy has been observed when it might keep damaged cells alive and supporting tumor formation.
Mitotic catastrophes are the result of mitotic cell division failure. A number of harmful assaults can induce genetic mutation in DNA which further leads dysfunction of cell cycle regulation and formation of multiple spindles; giant cell formation is the results of dysregulated mitotic cell division. This is also a form of cell suicide. The continuous division of these cell may leads to polyploidy and aneuploidy but death occurs very slow either apoptotically and necrotically. Mitotic catastrophe normally caused due to the accumulated of defects by dysfunction of cell cycle check points. However mitotic catastrophe occurs in those cells which compromised p53 function, because of p53 is the guardian of cell and regulates G1 and G2 check point. Mitotic catastrophe can be induced as consequence of premature entry of cell into mitosis with unpaired DNA damage due to compromised G2 check points. It can cause by abnormal duplication/division of centromere as these are crucial for the spindle pole formed during mitosis and for acute chromosome segregation in daughter cells. The stresses beyond threshold capacity enforced cell to die either classical cell death or non-classical cell death. However if the intensity of stresses are under threshold limit the cells will respond and DNA repair mechanism functions properly (Lindquist 1986). The stress response was first discovered as puffs of polytene chromosomes extracted from salivary gland of Drosophilla melanogaster larvae that have been given heat shock or chemicals (Ritossa et al 1962). He was Ritossa1962 who first visualized puffing in salivary gland chromosomes of D. melanogaster larval and concluded that the puffing is due to stressor (either heat shock or chemical). The hsps are super family of highly conserved group of protein expressed in almost all eukaryotic cells. In normal physiological condition Hsps express constitutively whilist pathological situation its expression induces many folds. Up regulation of Hsps expression correlates with evolution of thermo tolerance, a transient resistance to severe stress stimulates by below threshold stressors (Gerner and Schneider 1975). A number of other protein damaging stimuli can induce similar response to protect cell from further stressor induced cell death. (Landry et al 1982, Li et al 1982, Subjek et al 1982, Lindquist 1986). Misleading posttranscriptional modification results in the generation of misfolded and damaged protein which is the most common features of stressors. Further, the accumulation of abnormally folded and damaged protein induces the transcriptional activation of heat response genes as a result more expression of Hsps, (Ananthan et al 1986). HSPs acts as molecular chaperon in normal physiogical situations are responsible for proper folding of nasal polypeptide chain, for protein translocation and maintaining multi-protein complex in active conformations. Upon the exposure of stressors Hsps block the formation of protein aggregation and contribute refolding of misfolded and denature protein or target them for proteasome mediated degradation pathways (Gething and Sambrook 1992, Hartl et al 1992, Hartl and Mortin 1995, Bukau and Harwich 1998). The chaperon function of Hsp contributes to the restoration of cellular homeostasis after stressor and development of cytoprotective mechanism against phototoxic damage. The level of protection provided by Hsps is depend on the amount of Hsps that present in cellular pool after initial stress response and Cell death will still occur if levels of protein damage caused by sever stressors ( Mosser and Morimoto 2004).
Regulation of Hsps at transcriptional level
Stresses induce expression of heat shock protein mRNA by inhibiting transcription of others genes. In all respect, agent that can induce Hsps expressions do so through activation of specific transcription factor commonly known as heat shock factor1 (HSF1).
HSF family includes (HSF1, HSF2, and HSF4 in human, HSF1, HSF2, HSF3 and HSF4 in chicken and mouse) respectively, exhibit unique and overlapping functions, expressed in a tissue specific manner are responsible for a number of post translation modifications by increasing level of many cellular proteins [Reviewed a see Akerfelt, M. et al 2007, Akerfelt, M. et al 2010, Anckar, J. et al 2007, Bjork, J.K. et al 2010, Fujimoto, M. et al 2010]. HSF1 consist functional domains but most conserved among which is N-terminal DNA-binding domain (DBD). Different stressors/heat shock induces HSF1 oligomerization near DBD and form trimer from monomeric HSF1 and thereafter leads its posttranslational modulation. Upon trimerization/oligomerizaton HSF1 form hepted repeats of HR-A/B. The assembly of HSF1 trimer is prevented through another hepted repeat domains HR-C in normal physiological conditions [Wu, C. et al., 1995, Rabindran, S.K. 1993], which bind to HR-A/B and regulates HSF1 in monomeric conformation. The loss of HR-C domain induces continuous stimulation of HSF1 which may be corresponding to HSF4 that is difficult in HSF4, which has a constitutive ability of DNA binding [Wu, C. et. al., 1995]. HSF2 and HSF3 upon activation form dimmer which are responsible for binding to HSE (heat response element). Binding occurs in the form of penta-nucleotide motif "NGAAN" which consist of many inverted repeats of nucleotides (NGAAN). On the basis of HSF structure and architecture family member shows different binding pattern, thereby providing a board range of opportunity in the regulation of target genes (Fujimoto, M. et al 2008, Takemori, Y. et al 2009, Sakurai, H. et al., 2010). Therefore, HSFs can perform both function activator as well as repressor that depend upon situation of targeted genes [1-3]. Further, there is a cross talk among HSFs which combination provides possibilities for different post translational modification a fine correlation in the expression of HSF target genes. Furthermore HSF1, and HSF2 can assemble in the form of heterodimers in which HSF1 transiently modulate the activity of domain and protein candidate the HSFs family, HSF, and target genes [He, H. et al. 2003, Loison, F. 2006, Ostling, P. et al 2007, Sandqvist, A.et al 2009, Ahlskog, J.K. et al., 2010]. Stressor induced activation of HSF1 and HSF4 stimulates stress specific modification of histones through the requirement of chromatin remodulers SW1/SNF [Sullivan EK, et. al., 2001, Tu N, et al., 2006]. Therefore stressor activated HSF1 can modulate constitutive heterochromatin form to a transcription competent form. In the same way HSF2 has an epigenic role in hsps70 gene euchromatinization. During mitotic cell division HSFs interact with condensin protein and prevent chromatin condensation, the process termed as "Book marking" [Xing, H. et al. 2005]. Although HSF are responsible for Stress response and have several non-overlapping functions which is crucial in cancer therapeutics. HSFs can regulate proliferation differentiation asymmetric division and survival through activation/phosphorylation and deactivation hypophorylation their target genes. Among all HSFs, HSF1 is the master transcriptional factor of cellular response to variety of stress, maternal factor found in oocytes and play vital role in oogenesis preimplantaion development controlling the expression of Hsps, hsp70 mainly. HSF1 is essential for brain development and maintenance of germ cell, ciliated cell and immune cells by regulating hsp and non-hsp target genes (Nakai, A. et al 2000, Izu, H.et al 2004, Wang, G. et al 2004, Takaki, E. et al 2006, Inouye, S. et al 2004, Zheng, H. et al 2004). HSF1 play a vital role in initiation and maintenance of transformed phenotypes by facilitating tumor invasiveness in response to variable carcinogenic stimuli [Dai et al 2007]. Evidence from recent studies, HSF1 mice exhibit low rate of tumor by mutations of RAS protein and tumor suppressor p53 show prolonged survival. HSF1 help to retain transformed phenotype and tumorigenesis caused by human epidermal growth factor receptor-2 (HER-2), responsible for breast tumor progression. HSF1 knockdown studies suggest growth arrest and senescence of HER-2 expressed cells. HSF1 is known as regulator of transformed phenotype through established oncogenic cell line, breast cell line such as PHME, HME, HMLER, MCF-7, BT-474, MDAMD-231, and T47D. HSF1 is up regulated upon the exposure of injuries and malignancies whereas expression was observed at basal level in normal conditions. HSF1 itself have not oncogenic properties like RAS does because it helps in tumorigenesis and leads transformation itself and thus shows potential therapeutic challenging target. HSF1 regulates the core function of cellular pathways such as signal transduction, ribosome biogenesis, translation and glucose metabolism (Dai et. al. 2007). Cell proliferation and differentiation is regulated by HSF1 in normal/non-oncogenic type of cells/tissues but transformed phenotype of HSF1 can induce tumorigenesis. Evidences from yeast cell fission suggested that HSF1 a broad spectrum of biological functions, including protein folding and degradation, energy generation, protein trafficking and maintenance of cell integrity, transport of small molecules and cell signaling and transcription (Gallo G J et al 1993, Lecomte, S. et al., 2010, Westerheide, S.D. et al 2005). On the other hand murine HSF1 is highly dispensable for proliferation and survival of host, although evidence from HSF-/- mice exhibit defect in postnatal growth and placenta development whereas HSF1 in normal cell responsible for heat shock response which is more valuable to other cellular activities (Xiao, X. et al., 1999).
In normal mammalian system HSF1 regulate the expression of HSP genes but non-hsp genes also regulated during stress and differentiation. Cancer cell show high rate of proliferation due to elevated expression of HSF1 which further induces over expression of heat response gene or hsp gene. Over expressed heat response gene downstream signaling enhances expression of Hsps many fold, specifically hsp70 in tumor cells. There is no always correlation but highly expressed HSF1 induces over expression of hsp70. Evidence from knock down studies of hsp-/- were suggested that HSF1 down-regulates expression of hsp70 in many cancer cell lines. Hyperphosphorylation of HSF1 in many cancer cells and their cell line was detected maximum suggesting over expression of specific member of heat shock protein family hsp70 in particular. But the question is that why Hsps expression variable in cancer cells? Is it depending on hsp member? or tumor characteristic? HSF1 knock down studies confirmed down regulation of hsp70 expression in several tumor model (Meng, L. et al 2010).
HSF1 and p53
The first evidence of HSF1 implication comes from in vivo studies on p53 -/- mice in the development of spontaneous tumor. Progressive growth of lymphoma has been observed in mice lacking p53-/-, HSF1-/- mice rarely developed lymphoma, but quite yield of other carcinoma (89). Tumor suppressor p53 is suppressed by genetic mutations in many cancers and their cell line. HSF lacking mice genetically suppresses formation/growth of spontaneous tumor (Dae et al) in mice carrying dominant negative mutation of p53, whereas hsf +/+ and hsf+/- bearing dominant negative mutation of p53 develop broad range of tumor such as sarcomas, lymphomas, carcinoma (Dai, C. et al 2007). Further, it was confirmed using HSF1 knockdown mice or through siRNA in several mice and human cell lines (10)
Inhibition of HSF1 and cancer therapies
Evidences from knockdown studies in cancer cells demonstrate HSF1 inhibition should be effective strategy in cancer therapeutic. Several experimental reports on human HSF1 suggested variable p53 status, HER-2 expression, estrogen sensitivity and metastatic potential. Almost all tumors and their cell line affected significantly upon inhibition of HSF1. Rossi et al proposed ideal size target for siRNA mediated HSF1 silencing is 322-340 nucleotides. PSUPER-HSF1 vector potentially suppress HSF1 gene transcription as a result markedly increase in its sensitivity to hyperthermochemotherapy (combination of a cisplatin-treatment with heat shock), loading high rate of apoptosis of Hela cervical cancer cell line [Rossi, A. et al 2006]. These approaches of pharmacological targeting are attractive and interesting but still not well established for clinical use. In this contest modulating the activity of HSF1 by pharmacological agents will be a novel approach. A number of pharmacological inhibitors are available which can modulate the activation of serine/threonine kinases such as PKC and subsequent trimerization of HSF1. Among them some are in clinical trials such as quercetin, genestien. Synthetic benzylidenelactams, KN-alpha 437, and natural products stresgenenin-B has been observed to block HSPs expression but mode of action still in confusion (Akagawa, H. et. al., 1999). Quercetin acts as an anti-cancer agent (Limtrakul, P.et al., 2005, Jakubowicz-Gil, J. et. al. 2005, Shen, F. et al 1999) and cisplatin/tizofurin potent inhibiter of HSF1 and subsequently expression of HSPs. Cisplatin exhibit anti-proliferative and pro-apoptotic effect in HSF1 knockdown experiments in many cancers [Zanini C, et al., 2007]. The most potent inhibitor of HSF1 is triptalide, a diterpenetriepoxide form Tripterguim wilferdii which does not interfere in trimer formation, hyperphosporylation and DNA binding activity of HSF1 (Westerheide SD, et. al., 2006). Several cancer treatment approaches such as hsp90 inhibitors, Geldonmyin and Protexome inhibitor, Bortezomib stimulates proteotoxic effects by promoting pro-survival pathway but low cancer therapeutic value. Another two compound namely, NZ-28 and emunin their molecular action is not yet know but involved in post transcriptional modification of HSF1. Au et. al. identified another small molecule that can block granule formation of heat shock factor1 in hela cells and drastically inhibit HSF1 phosphorylation. The effect molecules were related to suppressed expression of HSF1 and subsequent expression of Hsp-90. Malaria drug quinacrine (QC) reported to block HSF1 dependent expression of hsp70 gene. The combination treatment of hsp90 inhibitor (17-DMAG) was revealed suppressed tumor growth in syngenic mouse model [Neznanov N, et. al., 2009].
Four major families of mammalian HSPs, classified into hsp90 hsp70 and hsp60 family on the basis of their size and small hsps includes hsp27. The study will focus on the role of hsp70 family in tumor development and apoptosis, which is more abundant than other and is probably well known in literature. The hsp70 family is evolutionary conserved group of proteins which express in all organisms. There are several isoform of hsp70, two in prokaryotes (seaton and vickery 1994) eight in mice and eleven in humans (R Tavaria et al). These proteins are highly abundant and consisting upto 2% of total cellular proteins (Anderson et al 1993). Hsp70 expressed in all cellular compartments such as cytosol, nucleus, mitochondria and ER lumen where they assisted proper folding and refolding of nascent polypeptide chain. Over expressed hsp70 in cytosol transported to nucleus and act protective transcriptional machinery under stress. In human/mice, there are two isoform of hsp70 that are hsp72/hsp73. Hsp73 constitutively expressed in all most all mammalian cells and referred as hsp73 and its expression increases many fold under-stresses. Further, over expression of hsp72 is mainly due to different stressors. Moreover, all HSPs expression increases during stress but expression of hsp72 most closely correlates with the development of thermo-tolerance (Li et al. 1982, Laszylo and Li 1985, Li 1985), however inhibition of HSPs, hsp70 expression or modulating its function renders cells extremely sensitive to heat shock (Johnston and kucey 1988, Riabowol et al. 1988).
Hsp70 has three distinct domain organizations, a highly conserved N-terminal ATPase domain (45kda), a substrate-binding domain C-terminal (25 K Da) and a regulatory domain EEVD motif at the extreame C-terminal end. ATP binding domain regulates substrate binding whereas EEVD sequence regulates ATPase activity of N-terminal domain, interaction with substrate and co-chaperon functions (Freeman et al. 1995, Burkholder et al. 1994, Buchberger et al. 1995). Hsp70 proteins specifically bind hydrophobic amino acid stretches with an optimum length of seven amino acid residue (Flynn et al. 1991). These residues are often supposed to similar regions that are often exposed when a peptide chain is incompletely unfolded (Rudiger et al. 1997). Hsp70 interact to substrate with their exposed residues to inhibit protein aggregation and assist proper folding of growing polypeptide chain (Bukau and Horwich 1998). In addition, hsp70 perform chaperone function by assisting proper folding/refolding of newly synthesized proteins in normal physiological condition. The whole process accelerated and/or regulated by ATP hydrolysis. Therefore, level of co-chaperon in cell can affect the role of hsp70. The binding of substrate is controlled by conformational changes that occurs during ATP binding and its hydrolysis (R Bukau and Harwich 1998, Hartland Hayer-Hartle 2002). ATP binding induce conformational change and exposed the peptide domain, thereby allowing substrate binding and release of product, in a highly specific manner.
Stress activated hsp70 and Apoptosis
Stressors up-regulate the expression of hsp70 many fold which can prevent protein aggregation and assist correct folding/refolding of denature proteins which are essential for removal of more severely damaged and misfolded proteins (Welch 1992, Nollen et al. 1999) and perform a protective role against adverse situations. However, deletion of ATPase domain results not any protective function against stress where as deletion of substrate binding domain abrogates its activity (Li et al. 1992). These results suggested that hsp70 is able to protect cells from heat shock/stress independent to its chaperon function. Evidences indicate that induction of thermotolerance protects cells from lethal injuries by inhibiting apoptosis and subsequently impeding cell death pathway, just through maintaining cellular homeostasis (Moser and Mortin 1992, Strasser and Anderson1995, Samali and Cotter 1996). Since then a number of studies have presented induced expression of hsp70 alone can protect cells from apoptosis, stimulated by heat shock, TNF-Î±, a ceramide and some cytotoxic drugs (Jaatella et al. 1992, Gabai et al. 1997, Mosser et al. 1997, Buzzard et al. 1998). Induced expression of hsp70 does not prevent heat induced apoptosis as effectively as induction of thermotolerance, but both have equal capacity to inhibit TNF-Î±- induced apoptosis (Buzzerd et al. 1998), supporting that full effect of apoptosis inhibition after some stresses, not other, need assistance of co-chaperones or other HSPs. Evidence from mutant forms of hsp70 suggested that substrate binding domain is sufficient to inhibit heat and TNF-Î±-induced apoptosis (Arichow et al). In contrast mosser and co-workers in a recent report demonstrated that ATPase domain and EEVD sequence are essential for hsp70 to inhibit stress induced apoptosis (Mosser et al. 2000). Further hsp70 has been known major player of apoptosis inhibition (R-Takayama et al 2003). Several other evidences have suggest a variety of apoptotic pathway and their distinct mechanism through which hsp70 prevent cells from a variety of harmful stresses. Over expressed hsp70 interact by its substrate binding domain with AIF and interferes with caspase independent apoptotic pathway. Therefore, hsp70 inhibit nuclear transport of AIF and AIF-related cell death mechanism in Apaf-1 null cells (Ravagnan et al. 2001, Gurbuxani et al. 2003). Inhibition of AIF-mediated apoptosis does not need ATPase activity of hsp70 (Ravagnan et al. 2001). Over expression of hsp70 can also inhibit heat shock induced apoptosis by blocking JNK and p38 activation (Gabai et al. 1997, Mosser et. al., 1997). Evidence from many other studies suggested that over expression of hsp70 suppressed JNK/SAPK phosphorylation through a variety of mechanisms which may also accelerate dephosphorylation (Meriin et al. 1999, Voloch et al. 1999, Yaglom et al. 2000, Gabai et al. 2000), or inhibit JNK/SAPK phosphorylation (park et al. 2001). Substrate binding domain of hsp70 induces JNK/SAPK dephosphorylation but not ATPase domain. However, the role of JNK/SAPK suppression in the inhibition of apoptosis has to be define by the finding that basal level expression of hsp70 does not prevent JNK/SAPK activation (Mosser et al. 1997 buzzerd et al. 1998, Jattela et al. 1998), and JNK may not be sufficient to induced apoptosis (Herr et al. 1999) and ATPase or EEVD null mutant block heat induced JNK/SAPK activation but not caspase activation or apoptosis (Mosser et al 2000). JNK/SAPK activation is essential for caspase-independent cell death but not necessary for caspase-dependent apoptosis. In conclusion HSPs knockout mice have added weight to the importance of hsp70 proteins in prevention of programmed cell death. Deletion of inducible hsp70 genes results in lack of ability to developed thermotolerance and mice are not protected from heat induced apoptosis (Huange et. al., 2001 and Van Molle et. al., 2002).
Hsp70 and Tumors
Over expression of hsp70 has been observed in some high grade tumors as compared to low grade tumors (Ciocca et al. 1993, Chant et al. 1995, kaur and Ralhan 1995, Lazaris et al. 1997, Athanassiadou et al. 1998, Nanbu et al. 1998). Highly expressed hsp70 inhibit breast tumor in similar way with resistance to combination chemotherapy, radiation, hyperthermia increased cell proliferation, poor differentiation lymph node metastasis and poor prognosis for disease free status and overall survival (Ciocca et al. 1993, Chan et al. 1995, Kaur and Ralhan 1995, Lazaris et al. 1995a/1995b, Ralhan and kaur 1995, Lazaris et al. 1997, Athanassiaoeu et. al., 1998, Nanbu et al. 1998). In addition, over expression of hsp70 enhances tumorigenic potential of rodent cells (JaaHela 1995, Volloch and Sherman 1999) and in transgenic mice hsp70 expression developed malignant T-cell lymphoma (Seo et al. 1996) whereas its inhibition through selective pharmacological inhibitors results inhibition of tumor cell proliferation and initiation of apoptotic events indicating hsp70 is needed for tumor cell survival (Wei et al. 1995, Nylandsted et al. 2000a/2000b, 2000). Therefore, targeting of hsp70 has been suggested as an anti-cancer therapeutics approach.
P53 and Apoptosis
P53 is known as the guardian of cell, act in response to a variety of phatho-physiological stresses to induce tumor growth inhibition and cell cycle arrest, DNA repair senescence and differentiation. It also helps in removal of old, damaged, infected and abnormal cell by controlling apoptosis, thereby preventing the development of cancer probability (Reviwed Fridman and Lowc 2003). Cancer cells exhibit a tendency to undergo a seemingly reversible cell cycle arrest. Not surprisingly p53 remain in the cell as an inactivated tumor suppressor in normal condition (Hussain and Harris 1998). Abrupt function of p53 leads cell cycle defects, genomic instability and not appropriate survival of host. Therefore, reactivation of p53 preferentially kills tumor cell and spare normal cell/tissue and thus may be attractive cancer therapeutic implication. P53 deficient cell line exhibit apoptotic features whereas p53 containing cell line does not show any apoptotic characteristics (Yonish- Rouach et al. 1991). This property of p53 was confirmed by studies with cell from p53 deficient mice, which show p53 was essential for stress induced apoptosis (Clarke et al. 1993, Lawe et al. 1993). Further, observation suggest that loss of apoptosis associated with tumor progression in p53 null mice indicated that apoptosis is necessary to tumor suppressor activity of p53 (Symonds et al 1994). P53exhibit its pro-apoptotic function mainly through transcriptional regulation of a growing number of target genes, a sequence specific DNA binding transcription factor (Ko and Prives 1996, Chao et al. 2000, Jimenez et al. 2000), transcriptional independent activity of p53 has also been described (Bates and Vousden 1999). P53 involved in both pathway(s) either intrinsic or extrinsic apoptotic pathway. In the intrinsic pathway, pathways, p53 up-regulate transcription of pro-apoptotic factors such as Bax, Bak and Bcl-xs (Miyashita and reed 1998), Puma (Nakano and Vousden 2001) and Naxo (oda et al 2000) as well Apaf-1 (Kannan et al 2001). In contrast, extrinsic apoptotic pathway is enhanced through transcriptional up-regulation of cell death receptors (TRAIL-R2) (Klu et al. 1999) and Fas (Owen- Schaub et. al., 1995) whilst enhancement of relocation of death receptor from Golgi to cell surface is an example of a transcription independent activity of p53 (Bennet et. al., 1998). Other transcriptional activity of p53 can affect mitochondrial events such as release of cyt-c. In addition recent report suggested that accumulation of p53 in cytosol following stress could directly activate Bax with similar kinetics to Bax activation by t-Bid (Chipu k et al. 2004). P53 has potential to arrest cell cycle progression. Progression induces transcription of p21 there is no single target essential to its ability to promote apoptosis such an example, Bax a well known target of p5