Matters of life and death: programmed cell death at Cold Spring Harbor.
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Biomedical subjects
S Cory
Publications and source records attributed to S Cory.
The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4.
Bcl-2 and close homologues such as Bcl-xL promote cell survival, while other relatives such as Bax antagonize this function. Since only the pro-survival family members possess a conserved N-terminal region denoted BH4, we have explored the role of this amphipathic helix for their survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. BH4 of Bcl-2 could be replaced by that of Bcl-x without perturbing function but not by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity was reduced by substitutions in two of ten conserved BH4 residues. Deletion of BH4 rendered Bcl-2 (and Bcl-xL) inactive but did not impair either Bcl-2 homodimerization or ability to bind to Bax or five other pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 lost the ability both to bind CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent apoptosis.
Amino Acid Sequence↗
Bim: a novel member of the Bcl-2 family that promotes apoptosis.
Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.
Amino Acid Sequence↗
The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry.
The Bcl-2 family of proteins regulate apoptosis, some antagonizing cell death and others facilitating it. It has recently been demonstrated that Bcl-2 not only inhibits apoptosis but also restrains cell cycle entry. We show here that these two functions can be genetically dissociated. Mutation of a tyrosine residue within the conserved N-terminal BH4 region had no effect on the ability of Bcl-2 or its closest homologs to enhance cell survival and did not prevent heterodimerization with death-enhancing family members Bax, Bak, Bad and Bik. Neither did this mutation override the growth-inhibitory effect of p53. However, on stimulation with cytokine or serum, starved quiescent cells expressing the mutant proteins re-entered the cell cycle much faster than those expressing comparable levels of wild-type proteins. When wild-type and Y28 mutant Bcl-2 were co-expressed, the mutant was dominant. Although R-Ras p23 has been reported to bind to Bcl-2, no interaction was detectable in transfected cells and R-Ras p23 did not interfere with the ability of Bcl-2 to inhibit apoptosis or cell cycle entry. These observations provide evidence that the anti-apoptotic function of Bcl-2 is mechanistically distinct from its inhibitory influence on cell cycle entry.
3T3 Cells↗
Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death.
Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, is an effective inhibitor of apoptotic cell death induced by many cytotoxic agents. Survival-promoting homologues of Bcl-2 include its close relative, Bcl-xL and the 19 kD protein encoded by the E1B gene of adenoviruses. Whether these proteins are functionally equivalent and whether they can antagonise all or only some pathways to apoptosis is unresolved. We have carried out a systematic comparison of Bcl-2, Bcl-xL and adenovirus E1B19kD activity, using several cell lines and a range of cytotoxic conditions. High levels of expression of each of these proteins inhibited apoptosis induced by growth factor deprivation or treatment with gamma-radiation, glucocorticoid and various cytotoxic drugs. In contrast, none of them could effectively counter apoptosis induced via the TNF receptor or Fas/APO-1 (CD95). Biochemical analysis revealed that all three proteins can associate with Bax and Bak, members of the Bcl-2 protein subfamily that can facilitate apoptosis. The results provide evidence that Bcl-2, Bcl-xL and adenovirus protein E1B19kD are indistinguishable in their ability to regulate the cell death effector machinery.
Adenovirus E1B Proteins↗
Lymphomas and plasmacytomas in transgenic mice involving bcl2, myc and v-abl.
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Deregulation of cell survival in lymphomagenesis.
Because normal lymphoid tissue homeostasis depends on a balance between cell proliferation and cell death, lymphomas can arise from mutations that interfere with the normal cell death process. We here discuss some circumstances in which lymphoid cell overexpression of a cell death antagonist, the Bcl2 protein, in E mu-bcl2 transgenic mice can contribute to the development of lymphomas and plasmacytomas.
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bcl-w, a novel member of the bcl-2 family, promotes cell survival.
The prototypic mammalian regulator of cell death is bcl-2, the oncogene implicated in the development of human follicular lymphoma. Several homologues of bcl-2 are now known. Using a PCR-based strategy we cloned a novel member of this gene family, denoted bcl-w. The gene, which is highly conserved between mouse and human, resides near the T-cell antigen receptor alpha gene within the central portion of mouse chromosome 14 and on human chromosome 14 at band q11. Enforced expression of bcl-w rendered lymphoid and myeloid cells refractory to several (but not all) cytotoxic conditions. Thus, like Bcl-2 and Bcl-x, the Bcl-w protein promotes cell survival, in contrast to other close homologues, Bax and Bak, which facilitate cell death. Comparison of the expected amino acid sequence of Bcl-w with that of these relatives helps to delineate residues likely to convey survival or anti-survival function. While expression of bcl-w was uncommon in B or T lymphoid cell lines, the mRNA was observed in almost all murine myeloid cell lines analysed and in a wide range of tissues. These findings suggest that bcl-w participates in the control of apoptosis in multiple cell types. Its functional similarity to bcl-2 also makes it an attractive candidate proto-oncogene.
Amino Acid Sequence↗
Progenitor tumours from Emu-bcl-2-myc transgenic mice have lymphomyeloid differentiation potential and reveal developmental differences in cell survival.
Mice expressing both a bcl-2 and a myc transgene within the B lymphoid cell compartment invariably develop novel immature haemopoietic tumours. The likely cell of origin of these tumours was identified by a common pattern of cell surface marker expression on a subset of cells comprising approximately 1% of normal mouse bone marrow. The bcl-2-myc tumour cells could be induced to differentiate into either B lymphocytes or macrophages in culture with certain cytokines and feeder cells. Analysis of their progression into the B lymphoid lineage revealed that Igk locus transcription can precede Igh as well as Igk rearrangement. Surprisingly, the undifferentiated tumour cells died rapidly in culture, even in the presence of multiple cytokines, but they proliferated on monolayers of stromal cells derived from haemopoietic tissues. Thus, even with Bcl-2 levels that protect more differentiated cells, these immature bi-potential progenitor cells require a stromal-induced signal for survival. These results provide insight into the process of lineage commitment and suggest new levels of control of cell survival during early steps in haemopoietic development.
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Lessons from bcl-2 transgenic mice for immunology, cancer biology and cell death research.
The protein product of the proto-oncogene bcl-2, originally discovered by virtue of its chromosomal translocation in human follicular centre B cell lymphoma, is a physiological inhibitor of programmed cell death, apoptosis. Initial studies in transgenic mice overexpressing Bcl-2 in B or T lymphocytes demonstrated that Bcl-2 can potently antagonise cell death induced by multiple independent signal transduction routes and can contribute to oncogenesis, particularly in combination with other oncogenes, like c-myc, that promote cell proliferation. Further investigations using crosses between bcl-2 transgenic mice and T cell receptor or immunoglobulin transgenic mice or mutant mice deficient in proper antigen receptor gene rearrangement demonstrated that Bcl-2 can only block death of cells that failed to receive a positive stimulus, "death by neglect', but not activation induced apoptosis. Collectively, these results provide evidence that distinct signalling pathways for apoptosis converge upon a common effector machinery where Bcl-2 acts as an antagonist, but that there also exists a mechanism that can either bypass the Bcl-2 checkpoint or override its protective function. These experimental data are reviewed here and discussed in context of current knowledge of lymphocyte differentiation, tumorigenesis and cell death regulation.
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Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.
Activation of the cell surface receptor Fas/APO-1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl-2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO-1-transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO-1-transduced apoptosis, but did not affect cell death induced by gamma-radiation or serum deprivation. Signalling through Fas/APO-1 did not down-regulate Bcl-2 or induce its antagonists Bax and Bcl-xS. In Fas/APO-1-deficient lpr mice, Bcl-2 transgenes markedly augmented the survival of antigen-activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto-reactive cells in the thymus). These data raise the possibility that Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.
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Regulation of lymphocyte survival by the bcl-2 gene family.
The control of cell survival is of central importance in tissues with high cell turnover such as the lymphoid system, and its disruption may be a critical step in tumorigenesis. Genes homologous to bcl-2, the oncogene implicated in human follicular lymphoma, play a key role in regulating physiologic cell death (apoptosis). Bcl-2 and its relatives bcl-x and bax encode intracellular membrane-bound proteins that share homology in three domains with a wider family of viral and cellular proteins. The Bcl-2 and Bcl-x proteins enhance the survival of lymphocytes and other cell types but do not promote their proliferation. High levels of Bax or of a smaller Bcl-x variant antagonize the survival function of Bcl-2. The mechanism by which Bcl-2 promotes cell survival remains unknown, but it appears to require association with Bax. Bcl-2 may combat the action of cysteine proteases thought to trigger apoptosis. Bcl-2 is not essential for embryogenesis or lymphoid development. However, upregulation of Bcl-2 appears to be the normal mechanism for positive selection of developing lymphocytes, and its continued expression is critical for survival of mature peripheral B and T cells. Constitutive expression of Bcl-2 does not abrogate deletion of self-reactive lymphocytes, nor disturb T lymphoid homeostasis; however, it substantially increases the pool of mature noncycling B cells. The risk of B lymphoid tumors is also enhanced, probably because Bcl-2 can countermand the apoptotic action of other oncoproteins such as Myc. Expression in tumors of bcl-2 and other cell survival genes may constitute a major barrier to the success of genotoxic cancer therapy.
Amino Acid Sequence↗
DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2.
The roles of p53 as an inducer and Bcl-2 as an inhibitor of apoptotic death were explored in lymphoid cells. Lymphocytes from p53-/- mice were radioresistant, but unexpectedly, cycling T lymphoma cells and mitogenically activated T lymphocytes from these animals underwent apoptosis after irradiation or genotoxic drug treatment. Hence, p53 is not the only mediator of apoptosis provoked by DNA damage. Irradiated p53-/- lymphoblasts expressing Bcl-2 were subject to growth arrest but resisted apoptosis. Their accumulation in G1 as well as G2 is suggestive of a p53-independent DNA-damage G1 checkpoint. Since Bcl-2 increased the clonogenic survival of the irradiated cells, expression of survival genes may pose a greater impediment to genotoxic cancer therapy than loss of p53.
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Insights from transgenic mice regarding the role of bcl-2 in normal and neoplastic lymphoid cells.
The bcl-2 gene was first discovered by molecular analysis of the 14;18 chromosome translocation which is the hallmark of most cases of human follicular lymphoma. To date, it is unique among proto-oncogenes because, rather than promoting cell proliferation, it fosters cell survival. This review summarizes the impact of constitutive bcl-2 expression on the development and function of lymphocytes as well as their malignant transformation. Expression of a bcl-2 transgene in the B lymphoid compartment profoundly perturbed homeostasis and, depending on the genetic background, predisposed to a severe autoimmune disease resembling human systemic lupus erythematosus. T lymphoid cells from bcl-2 transgenic mice were remarkably resistant to diverse cytotoxic agents. Nevertheless, T lymphoid homeostasis was unaffected and tolerance to self was maintained. Expression of high levels of Bcl-2 facilitated the development of B lymphoid tumours but at relatively low frequency and with long latency. Co-expression of myc and bcl-2, on the other hand, promoted the rapid onset of novel tumours which appeared to derive from a lympho-myeloid stem or progenitor cell. Introduction of the bcl-2 transgene into scid mice facilitated the survival and differentiation of pro-B but not pro-T cells, suggesting that a function necessary to supplement or complement the action of Bcl-2 is expressed later in the T than the B lineage. Crosses of the bcl-2 transgenic mice with p53-/- mice have addressed whether loss of p53 function and gain of bcl-2 function are synergistic for lymphoid cell survival.
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Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells.
Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient "compromised" bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor-derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.
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Bcl-2 expression promotes B- but not T-lymphoid development in scid mice.
Expression of antigen receptors is vital for the development of B and T lymphocytes. In mice with the scid mutation, which are unable to make productive rearrangements of their immunoglobulin and T-cell receptor (TCR) genes, lymphopoiesis aborts at an early stage. The death of the immature lymphocytes by apoptosis is postulated to result from a failure to receive a survival signal induced by receptor engagement. Consistent with this hypothesis, introduction of immunoglobulin or TCR transgenes into scid mice promoted an increase in B- or T-lymphoid cells, respectively. As the protein encoded by the bcl-2 gene can inhibit cell death, we tested whether lymphopoiesis could be rescued in scid mice by crossing in a bcl-2 transgene. Strikingly, the bcl-2/scid mice accumulated almost normal numbers of B-lymphoid cells which lacked surface immunoglobulin but expressed markers of maturity. T-cell development remained blocked. Introducing a TCR transgene enabled bcl-2/scid mice to develop normal numbers of CD4+8+ thymocytes even in the absence of immunological selection, suggesting that T cells become competent to respond to bcl-2 protein only after the TCR complex is displayed at the cell surface.
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Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.
To explore the role of bcl-2 in T-cell development, a bcl-2 transgene was introduced into mice expressing a T-cell receptor (TCR) transgene encoding reactivity for the mouse male antigen HY presented by the H-2Db class I antigen of the major histocompatibility complex (MHC). Normal thymic development is contingent on the ability of immature thymocytes to interact with self-MHC molecules presented by thymic stroma (positive selection). Thus, thymocyte numbers are low in female anti-HY TCR transgenic mice with a nonselecting (H-2Dd) background. Expression of bcl-2 inhibited the death of nonselectable thymocytes since, strikingly, female H-2Dd bcl-2/TCR transgenic mice developed normal numbers of CD4+CD8+ thymocytes, although these did not mature further into functional T cells. Hence, TCR-MHC interaction may induce positive selection through two signals, one which saves cells from death by increasing Bcl-2 synthesis and another which promotes maturation. Male H-2Db anti-HY TCR transgenic mice normally have a very small thymus, due to deletion of the self-reactive T cells. Expression of bcl-2 reduced the efficiency of deletion, since bcl-2/TCR transgenic male mice accumulated 4- to 6-fold more thymocytes than did TCR transgenic male littermates. Anti-HY TCR-expressing cells were also more numerous in the peripheral lymphoid tissues, but these cells expressed abnormally low levels of CD8 co-receptor and were not responsive to the HY antigen. Thus, although bcl-2 expression hampers the deletion of immature self-reactive cells in the thymus, self-tolerance is maintained.
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Apoptosis. Fascinating death factor.
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