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ORIGINAL ARTICLE
Year : 2006  |  Volume : 21  |  Issue : 1  |  Page : 1-11 Table of Contents   

Hypothetical Model for the Suppression of Stress Induced Apoptosis in Hematopoietic Stem Cells by Bcl-2 Mutants


1 Stem Cell Gene Therapy Research Group, Delhi, India
2 Dr. B. R. Ambedkar Centre for Biomedical Research, Delhi, India
3 INMAS, Delhi, India
4 Department of Biochemistry, V. P. Chest Institute, Delhi, India

Correspondence Address:
Gurudatta U Gangenahalli
Stem Cell Gene Therapy Research Group (INMAS), Lucknow Road, Timar Pur, Delhi-110054
India
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Source of Support: None, Conflict of Interest: None


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   Abstract 

Hematopoietic stem cells (HSC), which are responsible for maintaining continuous pool of blood cells, are being used for bone marrow transplantation (BMT). However, the programmed cell death/ apoptosis poses a serious problem for their optimum proliferation and differentiation after radio- and/ or chemotherapy. The role of Bcl-2 (B Cell Lymphoma) protein, a Bcl-2 family member, is well established in suppressing apoptosis of HSC on irradiation and serum withdrawal. The anti-apoptotic activity of Bcl-2 is regulated by inter- and intra-family homo-/ heterodimerization. Here we are proposing that the potential of Bcl-2 and its survival enhancing mutants, such as D34A and S70E, may be harnessed (gene therapy) to suppress the radiation and growth factor withdrawal induced apoptosis provided the neoplastic outcomes of these genes are regulated. The suggested hypothetical model is likely to be helpful in treating blood borne disorders and radiation injury through BMT.

Keywords: Hematopoiesis, bone marrow transplantation, Bcl-2 family, structure-function relationship, dosedependent overexpression


How to cite this article:
Gangenahalli GU, Verma YK, Singh VK, Gupta P, Chandra R, Sharma RK, Raj H G. Hypothetical Model for the Suppression of Stress Induced Apoptosis in Hematopoietic Stem Cells by Bcl-2 Mutants. Indian J Nucl Med 2006;21:1-11

How to cite this URL:
Gangenahalli GU, Verma YK, Singh VK, Gupta P, Chandra R, Sharma RK, Raj H G. Hypothetical Model for the Suppression of Stress Induced Apoptosis in Hematopoietic Stem Cells by Bcl-2 Mutants. Indian J Nucl Med [serial online] 2006 [cited 2019 Dec 6];21:1-11. Available from: http://www.ijnm.in/text.asp?2006/21/1/1/43433


   Introduction Top


Hematopoiesis is a process by which all the blood lineages are generated from the most primitive cells also known as hematopoietic stem cells. These, depending upon the signal, undergo either of its fate viz. proliferation, differentiation, homing and programmed cell death (apoptosis). The HSC reside in bone marrow of an adult individual whereas it is found in liver in children and yolk sac in embryos where hematopoiesis takes place respectively. The HSC are identified by the presence of various markers on its cell surface, which are CD34+, CD59+, Thy1+, CD38 low/- , c-kit -/low and lin - . However according to new information CD34 - cells are considered to be the most primitive, which before differentiation started to express CD34 antigen and may again undergo quiescency by suppressing the expression of CD34 antigen. Nowadays CD34 antigen is used as a basic marker for the purification and elutriation of HSC for various clinical purposes. CD34 + cells found its utility in blood borne disorders including lymphomas and radiation injury. The basic protocol that follows under these circumstances is the bone marrow transplantation. It is a process during which the primitive HSC are transplanted to reconstitute the normal hematopoiesis where this process is perturbed to reconstitute normal population of blood cells. The HSC as a source of BMT may be obtained from various sources including self (autologous), related donor (syngenic) and unrelated donor (allogenic). Under special circumstances where related/ unrelated HLA matched donor is unavailable the HSC of affected individual are aspirated, purified, purged and transplanted back along with various growth factors/ cytokines. The hyperproliferation and differentiation of HSC under such milieu limit the supply of growth factors, which should be constantly supplied. This is rather difficult due to expensiveness of growth factors. The decreasing concentration of growth factors makes HSC vulnerable to programmed cell death, which is among one of its fate. This lag in the generation of blood cells mediated immunity facilitates the opportunistic infection to sweeps in. This further complicates situation as affected individual now required to undergo high antibiotic dose regimen along with the growth factors.

The suppression of apoptosis by various techniques is likely to prevent the complication. However a chemical suppressor of apoptosis may not be a right choice due to its toxicity. Therefore a system is required which may permanently suppress apoptosis till optimum immunity is developed. Presently gene therapy approaches have been under trial stages to rapidly reconstitute normal hematopoiesis. It has been suggested that the Bcl-2 protein family members may have implications in suppressing growth factor deprivation and radiation induced apoptosis, which is regulated endogenously. Bcl-2 family consists of anti­apoptotic members (death suppressor) and pro-apoptotic (death inducer) members. The anti-apoptotic members include Bcl-2, Bcl-X L , Bcl-W, A1 and Mcl-1 etc and pro-apoptotic members include Bax, Bid, Bak, Bid, Harakiri, Bim, Bik, Bad and Bcl-X s etc. These members heterodimerize and homodimerize to nullify each other's action to regulate cellular homeostasis. The role of its anti-apoptotic members Bcl-2 is well documented in suppressing radiation and growth factors deprivation induced apoptosis in HSC and transgenic mice. The anti-apoptotic activity of Bcl-2 is regulated by various cellular proteins such as Raf-1 (Bcl-2 associating protein), BAG-1 (kinase), p53 binding protein (p 53 -BP2), PP2A (phosphatase) calcineurin, caspase-3 (apoptosis effecter protease), JNK (kinase), ubiquitin, PKC and ERK 1/2 (kinase) and NFAT. Bcl-2 protein is demarcated functionally into four Bcl-2 homology domains, one X domain, one transmembrane (TM) domain and one regulatory domain/ flexible loop domain (FLD). The TM domain is required for its localization to various cellular membranes such as mitochondria, endoplasmic reticulum and nuclear membrane where it regulates entry and exit of ions and neutralizes the mitochondrial pore forming protein Bax (pro-apoptotic). This neutralization is mediated by folding of BH3, BH2 and BH1 domains into a "BH3 receptor cleft"/ "active site" that interacts with amphipathic BH3 domain of pro-apoptotic proteins. The binding conformation of this cleft is affected by mutations in the regulatory domain of Bcl-2 whereas the presence of BH4 and X domain have no effect on heterodimerization however their presence is essential for the proper function of this protein.

In addition to Bcl-2 there are various known Bcl-2 mutants (D34A, S70E, ?FLD), which have cell survival enhancing effects. The anti-apoptotic activity of these mutants along with Bcl-2 may have gene therapeutic implications in suppressing HSC death to rapidly reconstitute the optimum immunity. However uncontrolled proliferation of these genes is a cause of concern, which may lead lymphoma in a long run. Therefore a fail-safe method should be in hand to regulate the expression of these genes. There are various inducible mammalian expression systems reported in which expression of a cloned gene could be controlled in a precise and regulated manner. The cell survival enhancing mutants under the controlled expression of these systems, in addition to Bcl-2, likely enhance the survival of HSC on stress induction. The expression of these genes may be switched "on" in genetically engineered HSC using the inducer till the optimum immunity is developed and switched "off" once this is achieved. The use of this strategy may find its usefulness under various clinical conditions where suppression of cellular death is the first concern for clinicians.


   Hematopoiesis and Hematopoietic Stem Cells Top


The highly orchestrated process of blood cell development and homeostasis is termed "hematopoiesis." Understanding the biology of HSC as well as hematopoiesis is important to develop improved treatments for hematological malignancies, congenital disorders, chemotherapy-related cytopenias and blood and marrow transplants. The HSC maintain continuous supply of different blood lineages by their self-renewal and differentiation. Depending upon the signal an HSC may have different fates including self-renewal, differentiation into variety of specialized cells, mobilization out of the bone marrow into circulating blood and programmed cell death (apoptosis) [1] . Hitherto there appears to be two kinds of HSC identified, long­term culture inititating cells (LTCIC) and short-term culture initiating cells (STCIC). If bone marrow cells from the transplanted mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its hematopoietic system over some months, they are considered to be LTCIC that are capable of self-renewal. Other cells from bone marrow can immediately regenerate all the different types of blood cells, but under normal circumstances cannot renew themselves over the long term, these are referred to as STCIC [2] . Hematopoietic progenitor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. They are capable of proliferating but have a limited capacity to differentiate into more than one cell type. Nowadays HSC are identified and purified for clinical purposes by the presence of various surface markers viz. CD34+, CD59+, Thy1 + , CD38 low/- , c-kit -/low , lin - . These markers exist in their undifferentiated state in in vivo and in vitro. As these cells begin to develop into distinct cell lineages the cell surface markers are no longer identified [3],[4] .

Although its precise function is still unknown, the pattern of expression of CD34 suggests that it plays a significant role in early hematopoiesis [5] . The theory of CD34 being the most primitive HSC marker, however, has recently been challenged. Osawa et al. first demonstrated that murine HSC could be CD34 negative. In addition, a low level of engraftment and hematopoietic capacity has been demonstrated in human CD34­cells [6] . Transplantation studies also showed repopulating activity in a CD34- cell population in fetal sheep [7] . Additionally, studies have shown that both murine and human CD34+ cells may be derived from CD34- cells [8],[9] . Collectively, these reports suggest the possibility that HSC may be CD34+ or CD34- and selection of cells expressing CD34 might result in exclusion of more primitive stem cells. Nevertheless, almost all clinical and experimental protocols including ex vivo culture, gene therapy, and HSC transplantation are currently designed for cell populations enriched for CD34+ cells.


   Regulation of Hematopoietic Stem Cell Populations Top


The number of cells in the bone marrow and blood is regulated by intricate signaling responsible for self-renewal, differentiation, homing and apoptotic death [1] . Apoptosis is the process of programmed cell death that leads cells to self­destruct when they are unneeded or detrimental. If there are too few HSC in the body, more cells divide and boost the numbers. If excess stem cells are injected into an animal, they simply wouldn't divide and undergo apoptosis. Excess numbers of stem cells in an HSC transplant actually seem to improve the likelihood and speed of engraftment, though there seems to be no rigorous identification of a mechanism for this empirical observation. Understanding the forces at play in HSC apoptosis is important to maintain or increase their numbers in culture. For example, without growth factors, supplied in the medium or through serum or other feeder layers of cells, HSCs undergo apoptosis which is the main cause for the failure of bone marrow transplantation [1] .

There are various strategy underway to suppress apoptosis of HSCs these includes cytokines, retinoids, antibodies to cell surface antigens and pharmacological reagents. Diferentiating cells can be rescued from apoptosis by re-adding IL-6 or by adding IL-3, M-CSF, G-CSF, SCF or IL­1. The differentiating leukemic cells can also be rescued from apoptosis by the tumor promoting phorbol ester 12-o­tetradecanoylphorbol 13-acetate (TPA). It rescues the differentiang cells from apoptosis by a different pathway than rescue with these cytokines. It is therefore possible to activate the normal physiological process of apoptosis in myeloid leukemic cells and this can be used to suppress leukemia even without induction of terminal induction. Interferon-y also inhibits induction of apoptosis in normal and leukemic myeloid cells by different apoptosis-inducing agents [10] . Studies with primary human myeloid leukemia cells have shown that GM­-CSF and IL-3 can protect these cells from patients with AML against induction of apoptosis by a chemotherapeutic compound such as the anthracyclin doxorubicin. It was also shown that IL-6 and other cytokines such as IL-1, IL-2 and IL­4 can suppress induction of apoptosis in normal and leukemic lymphoid cells by various cytotoxic agents [11] . However all these strategies have their limitations of providing only a temporary relief and constitutive concentration of these chemicals is required to sustain HSC in circulation. Whereas a high dose of these expensive chemicals may have toxic effects. To overcome these limitations a permanent solution may be gene therapeutics approach where an apoptosis suppressing gene may be introduced into HSC using transfection or transduction procedures (genetic engineering) which is likely to help in earlier immune reconstitution. In addition, these relatively apoptosis resistant HSC maay withstand the effect of post-transplantation irradaition to finish off the residiual leukemic cells.

Gene therapy of HSC has been tried with transduction/ transfection of genes such as IL-5Ra and c-kit, which regulates HSC number and cell fate decision [12],[13] . However no remarkable success has been achieved with these genes. Presently, Bcl-2 (B Cell Lymphoma) protein is being used in the research laboratories to make HSC radiation resistant, which may also survive under the decreasing growth factors conditions. Bcl-2 was originally identified as the oncogene activated by the characteristic t (14:18) translocation in non­Hodgkin's lymphoma [14] . Bcl-2 is a 25kD protein localized principally on the outer membranes of mitochondria, endoplasmic reticulum and nucleus. It is the firstly identified member of Bcl-2 family which inhibits cell death in a variety of stress conditions including deprivation of essential cytokines, heat shock, calcium ionophores, staurosporine, overdose of radiation and exposure to DNA damaging agents [15] . On the basis of cell death inhibition/ promotion, Bcl-2 family proteins are divided into anti- and pro-apoptotic members respectively. Four conserved domains have so far been identified in this family termed Bcl-2 homology (BH) domains viz. BH4, BH3, BH1 and BH2 [16] . Bcl-2 protein contains all four BH domains, BH4 (10-29 amino acids), BH3 (90-107 amino acids), BH1 (133-152 amino acids) and BH2 (184-199 amino acids). In addition to these it also possess one X domain (192-203 amino acids) and one regulatory/ flexible loop domain (FLD) (30-89 amino acids). In tertiary structure, Bcl-2 is folded into two central hydrophobic anti-parallel alpha helices i.e., α5 and α6, whereas α6, α7, α3 and α4 form flanking amphipathic helices creating a hydrophobic cleft. This cleft is essential for interaction of pro-apoptotic proteins such as Bax, Bik, Bid and Bim through their BH3 amphipathic α-helical domain [17] . There are multiple types of physical intra- and inter-family interactions displayed by Bcl-2. Intra-family interactions include homodimerization and hetrodimerization. The inter-family interactions occur with Raf-1 (Bcl-2 associating protein), BAG-1 (kinase) [18] , p 53 binding protein (p 53 -BP2) [19] , PP2A (phosphatase) [20] , calcineurin [21] , caspase-3 (apoptosis effecter protease), JNK (kinase) [22],[23],[24],[25] , ubiquitin [26] , PKC and ERK 1/2 (kinase) [27] and NFAT [28] .


   Regulation of Hematopoiesis and HSC Death by Bcl-2 Top


Functional studies of Bcl-2 have been carried out in a variety of cell lines, including those of lymphoid, myeloid, erythroid, neuronal, fibroblastoid and epitheloid origin. Overexpresion of Bcl-2 protects cell lines against growth factor withdrawal-induced apoptosis that required cytokines such as IL-2, IL-3, IL-4, IL-6, IL-7, GM-CSF, G-CSF, EPO or NGF for their proliferation and/ or survival cytokines [29],[30],[31],[32],[33],[34],[35] . In all these analysis, over-expression of Bcl-2 protected cells against apoptosis but was unable to promote cell proliferation. In growth factor independent cell lines, Bcl-2 antagonized apoptosis induced by exposure to broad range of cytotoxic conditions, such as gamma radiation, UV radiation, glucocorticoids, heat shock, cold shock, oxidative stress, calcium ionophores and phorbol ester [36],[37],[38],[39] .

To evaluate the impact of Bcl-2 ubiquitous overexpression within hematopoietic system, a mutant mouse was created which over-expresses Bcl-2 in hematopoietic system under the promoter of the vav gene (active throughout this compartment but rarely outside it). The vav-Bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. The mouse was shown to display increase in myeloid cells as well as a marked elevation in B and T lymphocytes. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and pre-progenitor cells. Thus, this study suggested that Bcl-2 has multiple effects on the hematopoietic system [40] .

Similar study was performed in the myeloid lineage of mutant op/op mice, which lack functional macrophage colony stimulating factor (MCSF/CSF-1) [41] . Over-expression of Bcl­2 allowed these cells to survive, rescued T-cell development in mice lacking IL-7 receptors [42] and promotes accumulation of B-cells that are unable to express surface immunoglobulin. Similarly, in rag-1 -/- and rag -/- mice [43] it enhances the survival of those T-cells, which express antigen receptors that are unable to interact with major histocompatibility complex molecules (inability to undergo positive selection) [44] . These results demonstrate that the absence of positive signals from neighboring cells trigger, in lymphoid and myeloid cells at several developmental checkpoints, an intrinsic death program that can be inhibited by Bcl-2.

More results which consolidates the role of Bcl-2 in suppressing radiation induced apoptosis comes from the study of H2K-Bcl-2 transgenic mice that over-expressed Bcl-2 in all cells of the hemato-lymphoid system and have been used to assess the role of Bcl-2 in protecting cells of the hemato­lymphoid system from the consequences of ionizing radiation. Systemic over-expression of Bcl-2 protected the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-Bcl-2 transgenic mice has two components; an increase in the radioresistance of individual cells and size of HSC population. Bone marrow transplantation experiments showed that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation was not limited to the increased expression levels of Bcl-2 in transgenic mice; levels of endogenous Bcl-2 were found to be higher in lymphocyte populations that survive irradiation in wild-type mice [45] . H2K-Bcl-2 transgenic mice also showed increased number of HSC in bone marrow (2.4x wild type), but fewer of these cells are in the S/G 2 /M phases of the cell cycle (0.6x wild type). The HSC also displayed an increased plating efficiency in vitro and have an advantage following competitive reconstitution with wild-type HSC [46] . These studies helped to further clarify the role of Bcl-2 in the development and homeostasis of this compartment.


   Mechanism of Bcl-2 Anti-apoptotic Action Top


Bcl-2 blocks the generation of reactive oxygen species and maintains cellular redox potentials, suggesting that it operates in an anti-oxidant pathway. It also alters intracellular ion fluxes that occur during apoptosis including changes in the partitioning of Ca 2+ in the endoplasmic reticulum, nucleus and mitochondria [47] . Other effects of Bcl-2 on mitochondria include inhibition of a mitochondrial permeability transition, a pre-apoptotic event that leads mitochondrial membrane swelling and loss of membrane potential. Bcl-2 has been shown to regulate and maintain the transmembrane potential across mitochondrial membrane through permeability transition pore (PTP). Bcl-2 and its family members such as Bcl-X L and Bax form mitochondrial membrane pores that act as channels for ions or possibly other molecules. By structural analogy to the bacterial toxins, the core of Bcl-X L and Bcl-2 consists of two central alpha helices (α5 and (α6) of largely hydrophobic residues, which insert across lipid bilayers and contribute to the formation of a mitochondrial membrane pore [48] . Recombinant forms of both Bcl-X L and Bcl-2 form ion channels at low pH in planar lipid bilayer or synthetic lipid vesicles, which are cation selective at physiologic pH. Bax forms pores both at acidic and neutral pH, these pores have biophysical characteristics distinct from Bcl-2 channels displays a mild Cl­ selectivity in contrast to the mild K+ selectivity exhibited by Bcl-2. At physiological pH the channel-forming activity of Bax is inhibited by the presence of Bcl-2. Membrane pores formed by Bcl-2/Bcl-X L act to heterodimerize with Bax protein so that Bax dimmers (active form) may not be able to initiate apoptotic events at the mitochondria that would otherwise lead to the cytosolic release of apoptosis triggering factors such as cytochrome c [49] .

Since Bcl-2 is present on outer membrane of endoplasmic reticulum (ER), many roles have been assigned to it that helps in regulating the process of apoptosis through ER. One potential model for this hypothesize that presence of Bcl-2 on ER sequesters "activator" BH3 only proteins, preventing them from interacting with Bax. "Activator" BH3 only proteins are displaced by "sensitizer" BH3 only proteins, freeing them to activate Bax, which translocates to mitochondria and induce cytochrome c release. The other model depicts that the ER lumen that serves as a source of calcium ions, releases them on inositol triphosphate (InsP3) receptor engagement with its ligand. The resulting elevation of cytosolic calcium directly mediates loss of mitochondrial membrane potential through increased uptake of calcium ions into the mitochondrial matrix, a process that is enhanced by permeabilization of the outer mitochondrial membrane by tBid. Indirectly Ca2+ activates calcineurin (phosphatase), which in turn activates Bad. Bcl-2 located on the ER membranes interferes with calcium mediated apoptotic signals, either by decreasing ER luminal calcium concentration or by docking calcineurin to InsP3 receptors, thereby inhibition InsP3-mediated calcium release and calcineurin mediated de­phosphorylation of phosphorylated Bad [50] . In T-cells, Bcl-2 inhibits T-cell receptor (TCR)-mediated activation of NFAT and induction of IL-2 expression. This inhibits cell cycle entry into S phase by delaying G0/G1 transition and subsequently TCR­activation-induced apoptosis. It has been suggested that Bcl-2 inhibits NFAT activation and entry into the nucleus through calcineurin by sequestering it to intracellular membranes. This raises the possibility that increased expression of Bcl-2 also plays a part in feedback loop that attenuates InsP3-mediated calcium signals and thereby controls T-cell proliferation while maintaining cell survival [50] .


   Structure Function Relationship of Bcl-2 Top


Bcl-2 is a 239 amino acid long protein, its BH1 and BH2 domains along with BH3 domain form a solvent accessible hydrophobic "BH3 receptor cleft"/ "active site" essential for heterodimerization with pro-apoptotic proteins [51],[52] [Figure 1].

The mutational data in Bcl-X L suggests a lock and key type interaction wherein BH1 and BH2 domains within the anti-apoptotic protein combine to act as a receptor for BH3 domain presented by the pro-apoptotic family members. Structurally it consists of 8 α-helices, the two core helices viz., α5 and α6 in Bcl-2α form two central hydrophobic anti­parallel channels in the mitochondria, endoplasmic reticulum and nuclear membrane similar to the bacterial toxin regulating Ca2+ flux and p 53-BP2 translocation across nucleus. These helices insert perpendicularly across the lipid bilayer opening like an umbrella, while the surrounding amphipathic helices fold upwards and rest on top of the membrane. Consistent with this idea removal of α5 and α6 from Bcl-2 prevents channel formation in vitro and abolishes its anti-apoptotic effect in cell [Figure 2]. The helices α3, α4, α6 and α7 encompassing BH4 and BH3 domains, form flanking amphipathic helices creating a hydrophobic cleft for hetrodimerization with pro­apoptotic proteins.

By analogy to Bcl-X L structural detail and interaction with Bak peptide, studies have shown that BH1 and BH2 domains in Bcl-2 are essential for its co-immunoprecipitation with Bax and for prolongation of cell survival. It has been shown that the residues viz. G145, R146 and W188 form an active site in Bcl-2 and its structural homologs [55] . Substitution of G145 (in BH1 domain) and W188 (in BH2 domain) with Ala disrupts the pore forming ability of Bcl-2 protein and completely abrogates its heterodimerization and consequently death repressor activity in IL-3 deprivation, gamma irradiation and glucocorticoid-induced apoptosis [56],[57] . Similarly, the substitution of G138A (G145 in Bcl-2) and R139Q (R146 in Bcl­2) in Bcl-X L alters the accessibility and binding properties of BH3 receptor cleft of Bcl-X L to pro-apoptotic proteins. The computer simulated Bcl-2 protein to Bax's BH3 domain docking has revealed that active site residue R146 of Bcl­ 2 forms hydrogen bonds with D68 and D71 of Bax peptide. Similar interactions were observed in Bcl-X L and Bak peptide complex, where interaction between D83 of Bak and R139 of Bcl-X L stabilizes the complex formation. The D83 residue, which is completely conserved within Bcl-2 family, when substituted with A in Bak peptide markedly, reduces the binding of peptide to Bcl-X L . Moreover conserved R139 mutation to E in Bcl-X L inhibits its anti-apoptotic activity and binding to Bak protein [58] . Bax mutant D68A has been shown to retain the ability to homodimerize but failed to interact with Bcl-2 as determined by yeast two-hybrid assays and co-immunoprecipitation analysis. The co-expression of wild type Bcl-2 with Bax mutant, D68A, rescues cells from apoptosis thus indicates the importance of D68 in heterodimerization interaction and induction of apoptosis by inhibiting Bcl-2 cell survival potential [59] . The other active site residue in Bcl-2, W188, docks with K64 of Bax peptide whereas small G145 provide the space to accommodate D68 within the groove formed by G145 and R146. Therefore, it has been suggested that any other amino acid in place of G145 would sterically inhibit the entry of D68 required to stably heterodimerize Bax protein with Bcl-2 [55] . These studies suggest that maintenance of structural integrity of BH3 receptor cleft/ active site is of utmost importance for Bcl-2 to heterodimerize with pro-apoptotic proteins.


   Post-Translational Regulation of Bcl-2 Top


The FLD of Bcl-2 lying in between BH4 and BH3 domains lack a defined structure and is not conserved among Bcl-2 family members. FLD deleted mutant of Bcl-X L and Bcl-2 displayed enhanced ability to inhibit apoptosis without impairing their heterodimerization with pro-apoptotic proteins [60] . FLD deleted mutant also displayed a quantitative difference in its ability to inhibit apoptosis. Full length Bcl-2 was unable to prevent anti-IgM induced cell death of the immature B cell line WEHI-231. In contrast, the mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the mutant compared with full length Bcl-2 [61] . By using two-dimensional peptide mapping and sequencing three residues (S70, S87 and T69) within FLD of Bcl-2 were found to be phosphorylated in response to microtubule damaging agent (paclitaxel), which also arrest T­-cells at G2/M. Changing these sites to A conferred more anti­apoptotic activity on Bcl-2 following physiological death signals as well as paclitaxel treatment indicating that hyper­phosphorylation is inactivating [62],[63] .

On the contrary, a single phosphorylation of Bcl-2 at S70 is essential for its full and potent cell survival activity. Bcl-2 protein gets phosphorylated by IL-3, PKC and erythropoietin on receiving signal in the normal conditions to maintain cellular homeostasis [64] . The S70A Bcl-2 mutant fails to be phosphorylated after IL-3 or Bryo stimulation and is unable to support prolonged cell survival either upon IL-3 deprivation or etoposide treatment. In contrast a S70E substitution suppresses the etoposide-induced apoptosis more potently than wild type Bcl-2 in the absence of IL-3. JNK-1 (SAK) was found out to be latently activated on IL-3 withdrawal, which phosphorylates Bcl-2 at S70 and co-localizes with it [65] . This data suggests that JNK-1 is the potent Bcl-2 kinase which upregulates/ downregulates Bcl-2 activity by phosphorylation/ hyper­phosphorylation respectively depending upon the cell survival/ death signal.

The phosphorylation of Bcl-2 has been a dynamic process, which involves both kinase and phosphatases, and a mechanism likely exists to rapidly and reversibly regulate its activity and functional consequences. [66],[67],[68],[69],[70] . Studies have revealed that PP2A is a physiologic Bcl-2 phosphatase since okadic acid and sphingolipid-ceramide sensitize PP2A and co-localize with Bcl­2 at the mitochondrial membrane [71],[72] . The molecular interaction of Bcl-2 and Bcl-X L with caspase 3 (Cysteine rich Aspartyl Proteases) affects their anti-apoptotic induction by cleaving Bcl-2 at D34 and Bcl-X L at 64. Caspases recognize tetra-peptide motifs and cleave their substrates on the carboxyl side of an Asp residue (P1 position). Individual caspases have distinct substrate specificity because they recognize different amino acids at three residues directly N-terminal of the Aspartate (P2, P3 and P4 positions) [73] . The caspase-3 mediated cleavage converts anti-apoptotic Bcl-2 into a Bax like pro-apoptotic protein that heterodimerizes with wild type Bcl-2 and induces apoptosis [74] . These mechanism, dephosphorylation and degradation, exist to downregulate the Bcl-2 function when it is not required to maintain the cellular homeostasis.


   In Vitro Mutagenesis Studies of Bcl-2 Top


As discussed earlier the point mutations G145A and W188A, in BH1 and BH2 domains respectively, severely impair Bcl-2 cell survival function. In addition anti-apoptotic activity is lost as a result of deletion of the BH4 region. Alterations in the corresponding regions within Bcl-X L [75] had similar consequences. Point mutations of highly conserved residues in BH4 and outside the BH domains generally had no effect on anti-apoptotic activity. The exceptions known to date are the mutation of non-conserved Serine 70 residue, which abolishes survival function and that deletion of FLD, which increases the cell survival activity of Bcl-2 [76] . Various Bcl-2 mutants having positive/ negative effect over its cell death suppressing activity are listed in the [Table 1] . However there are mutations in Bcl-2, which have insignificant effect on its function, are not listed.

On the basis of these studies and knowledge about Bcl-2 mutants we are proposing a strategy where over-expression of Bcl-2 and its growth enhancing mutants may be used to suppress the radiation and serum deprivation induced apoptosis. However there is a risk of leukemia generation when HSC started to over-proliferate under the influence of Bcl-2 over-expression, therefore, a fail-safe method is required so as to control the proliferation of genetically engineered HSC till the optimum immunity is developed and switch "off" the expression of Bcl-2. There are various inducible systems described in literature where the regulation of a cloned gene may be precisely regulated under the influence of inducer for example, lacZ [77] , Cre/loxP [78] , and Tet-On system [79] . In Tet-On system optimum overexpression of a cloned gene may be achieved by inducer such as doxycycline, the expression is switched "off" simply by withdrawing the inducer. To validate our strategy we have obtained few preliminary results which shows that the over-expression of Bcl-2 can really be controlled [Figure 3], which could potentially suppress the radiation induced apoptosis in HSC such as KG-1a. We have standardized the optimum dose of inducer for the Bcl-2 over­expression in KG-1a cells, which was found to be 1 ug/ml.

We generated two apoptosis suppressing point mutants (D34A and S70E) of Bcl-2 using cell based mutagenesis system [80] . The KG-1a cells were transfected with these mutants and then irradiated at lethal doses of gamma radiation [Figure 4]. We measured the suppression of apoptosis by using a caspase-9 detection kit (calbiochem). The caspase-9 is activated downstream in the Bcl-2 family mediated apoptosis. We found out that there was suppression of caspase-9 activation to the extent of 39% and 63% by Bcl-2 mutants D34A and S70E respectively in comparison to wild type Bcl-2 [81] . This study shows that Bcl-2 and its mutants possess the potential to suppress the radiation-induced apoptosis if their neoplastic outcomes could be controlled.


   Discussion Top


HSC has the potential to treat hematological malignancies through gene therapy, however, gene therapeutics finds hurdles of in vitro culturing and in vivo leukemia formation due to programmed cell death and constitutive expression of therapeutic gene respectively. Bcl-2 has shown potential to suppress serum-deprived and radiation induced apoptosis in vitro and in vivo. Our preliminary results showed that Bcl-2 and its mutants might be used under controlled induction (using doxycycline) for suppression of apoptosis. The hypothetical model, as suggested, is likely to suppress the stress-induced apoptosis, which is consolidated by our preliminary results. However, the regulation of HSC death may not be the most efficient method for the successful immune generation, therefore, HSC specific genes affecting proliferation (c-kit) [82] , adhesion/ homing (CD34) [83] and lineage generation (PU.1) [84] may also be considered for the genetic manipulation to provide rapid proliferation and lineage generation under the controlled induction. Still there are many unknown signals regulating the HSC fate, improvement may likely occur by the identification of new factors that will have the desired characteristics of preventing HSC death. The identification of factors involves experimental characterization or through recognition of sequence motifs in expressed sequence tag (EST) database, cDNA libraries and genome project [1] . In addition, in silico tools will speed up the identification of factors, which may maintain and expand HSC without compromising on their pluripotency.


   Acknowledgement Top


We are thankful to Director, Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timar Pur, Delhi-110054 and Dr Vani Brahamchari of Dr. B. R. Ambedkar Centre for Biomedical Research (ACBR), University of Delhi, Delhi­110007, India, for their support. Authors also thank Mr. J. S. Adhikari for analyzing the flow cytometry data. Mr. Yogesh Kr. Verma in particular thanks Council for Scientific and Industrial Research (CSIR), India, for awarding research fellowship for PhD from ACBR.

 
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