BCL-2 Gene Family and the Regulation of Programmed Cell Death 1

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[CANCER RESEARCH (SUPPL.) 59, 1693s-1700s, April 1, 1999] BCL-2 Gene Family and the Regulation of Programmed Cell Death 1

Stanley J. Korsmeyer 2'3

Division of Molecular Oncology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

I hope he doesn't mind me saying this, but Dr. Stan Korsmeyer has achieved an enormous amount in a relatively short scientific career. You can take that whichever way you want to. Good, now I have landed myself in it. Stun Korsmeyer did his undergraduate degree in the University of Illinois and then did an M.D. at the same university and graduated in '76. Then, after doing an internship in UCSF Medical School, he came to the NIH where he was a clinical associate working in collaboration with Phil Leder and Tom Waldmann. There are some interesting parallels with Suzanne's career at this stage. Stan worked quite a lot on the structure of the immunoglobulin genes, their rearrangements in normal B cell development, and also studied aberrant rearrangements in leukemias. He set up his own lab in, I believe it was, '83 at NIH and then moved to Washington University School of Medicine at St. Louis where he became an associate professor in about 1986. The first pivotal contribution in relation to this prize, was his isolation, at the same time as others, of the bcl-2 oncogene at the breakpoint of a translocation between chromosomes 14 and 18 in B cell follicular lymphoma. Very shortly after Suzanne and colleagues published their seminal study showing that bcl-2 promotes and extends B cell survival, Stan showed essentially the same thing in transgenic mice where he overexpressed the bcl-2 oncogene in B cells and was able to show that B cells had extended survival and went on to develop lymphomas. So, a wonderful convergence of in vitro findings in cell culture from Suzanne's lab and Stan's finding in mice. Stan has continued over the years to make some very important contributions to our understanding of bcl-2 function and also of bcl-2 relatives, as you have already heard from Suzanne. One of the first things he showed was that bcl-2 may have a function in mitochondria, and this notion received a great deal of support from findings published last year by several groups. Using a very elegant set of biochemical approaches, he was able to show that bcl-2 is a member of a larger family and that these family members can interact with each other. As you have heard, some of them act to promote and some to inhibit apoptosis. So, we have a very interesting network of proteins here. By knocking out different members of these families, Stan was also able to show that these genes really do play vital roles in cell survival in whole animals, obviously, a very important thing to do. He continues to work on the function of the proteins, and it is remarkable to think that, while doing this, he also was able to show the important roles of Hox-11 and Mll both in normal development and in leukemias. Since 1992 Stan has been the Chief of the Division of Molecular Oncology at Washington University School of Medicine, St Louis and since 1993 has been an investigator of the Howard Hughes Medical Institute. In recognition of his pioneering work, Stan has already received a number of awards including the Ciba Drew Award in 1996, the GHA Clowes Award, American Association for Cancer Research in 1997 and the Bristol-Myers Squibb Award for distinguished achievements in cancer research, 1997. He was elected to the National Academy of Sciences in 1995. So, we look forward very much to hearing Stan's talk on the bcl-2 family and the regulation of cell death. Nicholas D. Hastie Medical Research Council Human Genetics Unit Western General Hospital Edinburgh, United Kingdom

Abstract homology clustered within four conserved regions termed BCL-2 homology 1 through 4 (BHI-4). These novel domains control the ability of these proteins The BCL-2 gene was identified at the chromosomal breakpoint of t(14; to dimerize and function. An amphipathic a helix, BH3, is of particular 18)-bearing human follicular B cell lymphomas. BCL-2 proved to block importance for the proapoptotic family members. BID and BAD represent an programmed cell death rather than promote proliferation. Transgenic mice evolving set of proapoptotic molecules, which bear sequence homology only at that overexpress Bcl-2 in the B cell lineage demonstrate extended cell survival BH3. They appear to reside more proximal in the pathway serving as death and progress to high-grade lymphomas. Thus, BCL-2 initiated a new cate- ligands. BAD connects upstream signal transduction paths with the BCL-2 gory of oncogenes, regulators of cell death. Bcl-2-deficient mice demonstrate family, modulating this checkpoint for apoptosis. In the presence of survival fnlminant apoptosis of lymphocytes, profound renal cell death and loss of factor interleukin-3, cells phosphorylate BAD on two serine residues. This melanocytes. BCL-2 protein duels with its counteracting twin, a partner inactivated BAD is held by the 14-3-3 protein, freeing BCL-XL and BCL-2 to known as BAX. When BAX is in excess, cells execute a death command; but, promote survival. Activation of BAX results in the initiation of apoptosis. when BCL-2 dominates, the program is inhibited and cells survive. Bax- Downstream events in this program include mitochondrial dysfunction, as deficient mice display cellular hyperplasia, confirming its role as a pro- well as Caspase activation. The pro- and antiapoptotic BCL-2 family mem- apoptotic molecule. An expanded family of BCL-2-related proteins shares bers represent central regulators in an evolutionarily conserved pathway of cell death. Aberrations in the BCL-2 family result in disordered homeostasis, Received 9/18/98; accepted 1/27/99. a pathogenic event in diseases, including cancer. 1 Presented at the "General Motors Cancer Research Foundation Twentieth Annual Scientific Conference: Developmental Biology and Cancer," June 9-10, 1998, Bethesda, MD. Introduction 2 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, Smith 758, Harvard Medical School, One Jimmy Fund Way, Boston, MA 02115. 3 Co-recipient of the Mott Prize along with Suzanne Cory, whose article can be found Programmed cell death plays an indispensable role in the develop- on pages 1685s-1692s of this Supplement. ment and maintenance of homeostasis within all multicellulax organ- 1693s

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. BCL-2 FAMILY REGULATES APOPTOSIS isms. Genetic and molecular analysis from nematodes to humans has chromosomal breakpoint. Aberrant immunoglobulin heavy chain indicated that the pathway of cellular suicide is highly conserved. rearrangements in t(14;18) lymphomas proved to be the chromo- Although the capacity to carry out apoptosis seems to be inherent in somal breakpoint and delivered a candidate oncogene, BCL-2, at all cells, the susceptibility to apoptosis varies markedly and is influ- 18q21 (1-3). BCL-2 is normally located on chromosome segment enced by external and cell-autonomous events. Considerable progress 18q21.3 in a telomere to centromere orientation. A molecular has been made in identifying the molecules that regulate the apoptotic consequence of the translocation is the movement of the BCL-2 pathway at each level. Of note, both positive and negative regulators, gene to the der (14) chromosome (Fig. 1). This places BCL-2 in the often encoded within the same family of proteins, characterize the same transcriptional orientation as the immunoglobulin heavy extracellular, cell surface, and intracellular steps. chain locus, giving rise to chimeric RNAs. However, translocation The t(14;18) Breakpoint of Human Follicular Lymphoma Pro- does not interrupt the protein-encoding region so that normal and vides the BCL-2 Gene. The t(14;18):(q32;q21) constitutes the most translocated alleles produce the same-sized, 25-kDa protein, t(14; common chromosomal translocation in human lymphoid malignan- 18)-bearing B cells have inappropriately elevated levels of the cies, being present in 85% of follicular and 20% of diffuse B-cell BCL-2-immunoglobulin fusion RNA and BCL-2 protein (4, 5). lymphomas. As a disease, follicular lymphoma provided many BCL-2-immunoglobulin Transgenic Mice Progress from Follic- clues concerning the ultimate function of the BCL-2 molecule. ular Hyperplasia to High-Grade Lymphoma. A stringent test of a Follicular lymphoma often presents as a low-grade malignancy gene's oncogenic capacity is to place it into the germline of mice composed of small resting igM/IgD B cells. Over time, conversion to observe its effects during the development of an entire organism. to an aggressive high-grade lymphoma with a diffuse large-cell Transgenic mice bearing a BCL-2-immunoglobulin minigene initially architecture frequently occurs in these patients. The location of the displayed a polyclonal follicular lymphoproliferation that selectively ex- immunoglobulin heavy chain locus at 14q32 and the B-cell phe- panded a small resting IgM/IgD B-cell population (Refs. 6, 7; Fig. 1). notype of this lymphoma provided the rationale for cloning the Cell cycle analysis confirmed that ~97% of the expanded B cells reside

Transgenic Construct Chromosome Chromosome I 18 I 14 J I II III P, P, J, EH Sj, Sv Cv, pA pA Translocated i ! P~2~: ~ ~,r ~ ~:~,~ .... ~;~im~:'ltI~tul~llml~;mwJ~ Bcl-2-Tg Genomic cDNA' ' Genomic 1 H23vs ,.~, Normal Sequence lOS_ Splenocytes - 0 - 0 Bcl-2 Transgenic - 9 - 9 Control Littermate

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! I V 10; 2 4 6 8 Days in Culture Fig. 1. Bcl-2-immunoglobulintransgenic mice. The Bcl-2-immunoglobulinminigene recapitulates the t(14;18) chrolnosomaltranslocation of follicular B-cell lymphoma.Gain of function Bcl-2 leads to B-cell expansion and an enlargedwhite pulp in the transgenic (left) versus control (right) spleen. B cells accumulate due to extendedcell survival (graph). 1694s

Table 10ncogene categories membrane blebbing, volume contraction, nuclear condensation, and Category 1: Growth and proliferation genes endonucleolytic cleavage of DNA, (15) termed apoptosis by Wyllie Transcription factors or signal transduction (17). By preventing the apoptotic demise of activated lymphocytes, Myc, Ras, Abl Gain of function BCL-2 enables the acquisition of additional genetic aberrations and Category II: tumor suppressor genes the emergence of monoclonal neoplasms. By repressing normal ap- Inhibit growth and proliferation optotic pathways, BCL-2 allows cells that sustain DNA damage to Rb, p53 Loss of function avoid a suicide response. Category 11I: programmed cell death genes Oncogenic events had concentrated on mechanisms of increased Antiapoptotic genes growth and proliferation. However, increased cell number, which Bcl-2 Gain of function violates normal homeostasis, could arise from either increased Proapoptotic genes proliferation or decreased death. BCL-2 provided the first example p53, Bax Loss of function of oncogenesis mediated by decreased cell death. Table 1 depicts two classes of oncogenes that regulate growth and proliferation (18). The first oncogenes discovered, category I, promote cell growth and proliferation. Most of these can be classified as transcription factors or molecules involved in signal transduction. In general, these genes contribute to cancer after an alteration resulting in a gain of function. They usually display an autosomal dominant mechanism, in which a single altered allele is sufficient to confer the effect. Category II represents the classic tumor suppressor genes that, in their wild-type form, inhibit growth and proliferation (19). A unique aspect of two experimentally proven ipO members, Rb and p53, is their frequent contribution to unchecked growth by loss of function. The effects of deregulated BCL-2 argue that it does not qualify as either a category I or II oncogene. Instead, BCL-2 seems to be the cardinal member of a new category Fig. 2. Susceptibility to PCD. The relative ratio of BCL2 to BAX determines the of oncogenes: regulators of PCD (Table 1). susceptibility to PCD. BAX, a Proapoptotic Member and Expansion of the Family. The first proapoptotic homologue, BAX, was identified by coim- in Go/Gv These recirculating B cells accumulate because of an extended survival rather than increased proliferation. Despite a 4-fold increment in resting B cells, BCL-2-immunoglobulin mice are initially quite healthy. The BCL-2 Family However, over time, these transgenics progress from indolent follicular hyperplasia to diffuse large-cell immunoblastic lymphoma. A long la- Anti-apoptotic tency period and progression from polyclonal hyperplasia to monoclonal high-grade malignancy is an indictment of secondary genetic abnormal- Mammalian BH4 BH3 BH1 BH2 TM BCL-2 ities. Approximately half of the high-grade tumors possess a c-myc translocation involving an immunoglobulin heavy-chain locus (8). These BCL-X L tumor cells have complemented an inherent survival advantage (bcl-2) BCL-W with a gene that promotes proliferation (myc). When bcl-2 transgenic MCL-1 mice were mated with myc transgenic mice, rapidly emerging undiffer- A1 entiated hematopoietic leukemia occurred, providing further testimony for the potent synergy of this particular oncogene combination (9). In NR-13 [Rxx-q--t~N-s addition to promoting cell cycle progression, myc has been shown to C. elegans CED-9 promote cell death (10). Thus, the overexpression of myc specifically benefits from the ability of bcl-2 to block cell death. The BCL-2-imInu- Pro-apoptotic noglobulin mice document the prospective importance of the t(14;18) in Mammalian setting the stage for lymphomagenesis and tumor progression. T-cell BAX lymphomas were also found in mice where bcl-2 was overexpressed by BAK the lck promoter (11). In this case, thymocytes and peripheral T cells also BOK exhibited enhanced cell survival (12, 13). Thus, prolonged cell survival can be a primary oncogenic event in both B and T cells. BCL-X S -~ ~ E} BCL-2 Blocks Apoptosis Initiating a New Category of Onco- BH3-only genes: Regulators of PCD. BCL-2 blocks cell death after a wide BID variety of stimuli. BCL-2 conferred a death-sparing effect to hema- BAD F'77"~~ topoietic cell lines after growth factor withdrawal, including the BIK --~ ...... E]- IL-3a-dependent early hematopoietic cell lines FDCP1, FL5.12, and BLK ~1~7"/~ {~- 32D (14-16). A detailed examination of the interference of BCL-2 with cell death revealed it does not alter the dose response to limiting HRK concentrations of IL-3 (16). Instead, BCL-2 blocked the plasma BIN F~ Fig. 3. Summary of antiapoptotic and proapoptotic BCL-2 family members. BCL-2 4 The abbreviations used are: IL-3, interleukin-3; PCD, programmed cell death. homology regions are indicated with similar shaded boxes (BH1-BH4). 1695s

Table 2 Bcl-2 -/- BCL-2 proteins and proved essential for proapoptotic function (31, B + T cell apoptosis: immunodeficiency 32, 33). The multidimensional nuclear magnetic resonance and X-ray Renal mesangial apoptosis: polycystic kidney crystallographic structure of a BCL-X L monomer indicated that Melanocyte death: hypopigmentation $ Primordial follicles: 9 infertility BH1-4 domains corresponded to o~-helices 1-7. The a-helices of BH1-3 are closely juxtaposed to form a hydrophobic pocket (34). Nuclear magnetic resonance analysis of a BAK peptide/BCL-XL interaction and a detailed mutational analysis of the BH3 amphipathic Table 3 Bax -/- c~2-helix of BAX indicates that BH3 forms critical interactions with Mild lymphoidhyperplasia BCL-2/BCL-XL (35). Moreover, the hydrophobic face of this am- Spermatogonia hyperptasia: ~ sterility Neuronal hyperplasia: trophic factor (NGFa) independence;protection from stroke phipathic helix is critical for its death-effector function (Ref. (33); Fig. 4). 1' Primordialfollicles: oocyte chemoresistance;no menopause Loss of Function Models Reveal Critical Roles for the BCL-2 Accelerated onset of tumors in the presenceof an oncogene:Bax as a tumor suppressor Family in Homeostasis. The normal developmental roles of BCL-2 "NGF, nerve growth factor. family members have been addressed by gene disruption. These loss of function models have proven most instructive in revealing the cell type specificity and singular roles for the BCL-2 members. munoprecipitation with the BCL-2 protein (20). BAX is a 21-kDa Newborn Bcl-2-/- knockout mice are viable, but the majority die protein that shares homology with BCL-2 clustered in conserved at a few weeks of age (Ref. (36); Table 2). They develop polycystic regions entitled BCL-2 homology domains, BH1-3. BAX het- kidney disease with marked dilation of proximal and distal tubules erodimerizes with BCL-2 and homodimerizes with itself. When and collecting ducts, resulting in renal failure (36-38). In the BAX was overexpressed in cells, apoptotic death in response to a normal fetal kidney, BCL-2 maintains cell survival during induc- death signal was enhanced, earning its designation as a death tive interactions between epithelium and mesenchyme. BCL-2-/- agonist. When BCL-2 was overexpressed, it heterodimerized with kidneys contain many fewer nephrons and greatly increased apop- BAX and death was repressed. Thus, the ratio of BCL-2 to BAX tosis within metanephric blastemas of metanephroi at embryonic serves as a rheostat to determine the susceptibility to apoptosis day 12 (39). Bcl-2-/- mice turn gray at 5-6 weeks of age, at the (Fig. 2). time of the second hair follicle cycle. The hypopigmentation of The family has further expanded to include the death antagonists Bcl-2-/- mice reflects decreased melanocyte survival. The BCL-2, BCL-X L (21), MCL-1 (22), and A1 (23), as well as the lymphoid organs, thymus, and spleen are initially normal in Bcl- proapoptotic molecules BAX, BCL-X s (21), and BAK (24-26). Con- 2-/- mice. Thymocyte development is normal, and B and T cells served domains BH1, BH2, and BH3 participate in the formation of undergo selection successfully. However, at 4-8 weeks of age, the various dimer pairs, as well as the regulation of cell death (Refs. (27 lymphoid organs undergo massive cell death and involution, show- and 28); Fig. 3). ing a failure to maintain homeostasis in both the B- and T-cell Mutational analysis of BCL-2 and BCL-X L identified key residues populations in the absence of BCL-2. within BH1 and BH2 domains required for both heterodimerization Bax-deficient mice represent the first knockout of a death- with BAX and repression of cell death (29). However, other BCL-XL promoting family member (Ref. (40); Table 3). Bax-/- mice mutants lost heterodimerization with BAX, but still retained some appear healthy, indicating that BAX is not essential for develop- death-repressor activity, suggesting that these two functions were ment of a viable organism. However, male Bax-/- mice are separable (30). An additional domain, BH3, had also been noted in infertile, and Bax-/- seminiferous tubules are markedly disorga-

Fig. 4. Three-dimensional model of the BH3 domain of BAX. Model of the molecular surface of BAX BH3. The surface is colored deep blue (23kBT) in the most positive regions and deep red (-21kBT) in the most negative, with linear inter- polation for values in between. Left, view of the hydrophobic surface of the amphipathic BH3 helix of BAX. Right, view of the polar surface of the amphipathic BH3 a-2 helix of BAX. Residues forming polar and hydrophobic surfaces are indicated.

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Normal Tissue autoimmune disease, whereas accelerated cell death can manifest as neurodegenerative disease, immunodeficiency syndromes, or infertil- (In) 1 ity (Fig. 5). Proapoptotic BAX as a Tumor Suppressor. Prolonged cell sur- J Homeostasis vival with resistance to apoptosis can be a primary oncogenic (Out) 1 event. This predicted that proapoptotic genes in the death pathway might contribute to oncogenesis through loss of function mutations (Table 1). Indeed, a principal contribution from the loss of p53 Diseases of Disordered Cell Death function is the elimination of a death pathway (43, 44). In select settings, stimuli that induce p53 result in the expression of Bax Proliferation 1 (45). Bax-deficient mice indicate that approximately half of certain p53-dependent cell deaths require BAX. Elimination of BAX Neurodegenerative blocks approximately half of chemotherapy-induced cell death Immunodeficiency Cancer (46). A transgenic model, TGT121 expresses a truncated T antigen Infertility Autoimmunity that inhibits Rb but leaves p53 intact, resulting in cell proliferation with counterbalancing cell death. The elimination of Bax in this 1 Death l model markedly decreased the apoptosis. Moreover, the TGT121 transgenic mice displayed a markedly accelerated progression to Fig. 5. Schematicrepresentation of normal tissue homeostasiswith balancedinput and output reactions. Primary aberrations in the cell death pathway that result in diminished malignancy on a Bax-deficient background (Ref. (47); Table 3). death can manifest as cancer or autoimmunity, whereas increased death can result in These experimental models argue that Bax can also be considered neurodegenerativedisease, immunodeficiency,or infertility. a tumor suppressor. Recently, mutations in BAX were noted in ~20% of human hema- nized with multinucleated giant cells, clusters of apoptotic germ topoietic malignancies studied (48). Approximately half were frame- cells, and lack of mature sperm. This follows a failure of normal shifts confined to a single mononucleotide (G) s tract (nt 114-121) postnatal death of spermatogonia, leading to an expansion of the that results in a stop codon and the loss of BAX protein. The others premeiotic 2N cell population. Thus, the failure of BAX-dependent represent point mutations within conserved BH1 and BH3 domains developmental deaths leads to organ failure and later paradoxic that alter dimerization and eliminate proapoptotic activity. Rampino et cell death during attempted meiosis (40). Bax-/- ovaries display al. (49) described the presence of frameshift mutations in the identical an accumulation of atrophic granulosa cells and excess primordial (G)8 tract of BAX in about 50% of human colon adenocarcinomas with follicles that failed to undergo apoptosis (41). Bax-/--sympa- the microsatellite mutator phenotype. Mutations of the proapoptotic thetic neurons do not reqmre nerve growth factor for survival, and BAX molecule within human malignancies support the role of BAX motor neurons survive disconnection from their targets after axo- as a tumor suppressor. tomy (42). The Bax-deficient mice separated the survival from BH3 Only Molecules as Death Ligands That Connect the anabolic action of neurotrophic factors. Despite the presence of BCL-2 Checkpoint to Signal Transduction. To extend the cell multiple BCL-2 family members within these neurons, their death death pathway, BCL-2 and BAX were used as bait in interactive by trophic factor deprivation was singularly dependent on BAX. cloning strategies. This provided a series of interacting molecules that Transgenic and knockout models of the Bcl-2 family indicate that bear sequence homology to BCL-2 principally within only the BH3 primary aberrations in the cell death pathway result in disordered domain. These now include BIK (50, 51), BID (52), BAD (53, 54), homeostasis and disease. Decreased cell death can result in cancer or BIM (55), HRK (56), and BLK (Ref. (57); Fig. 3). Mutagenesis of

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Fig. 6. Dual impact of lL-3-induced phosphorylation of BAD on two serine sites by distinct kinase pathways. The phosphorylated BAD is se- N Inactive questered by 14-3-3 in the cytosol as the inactive form, which releases BCL-X L to promote survival. Only nonphosphorylated BAD can heterodimerize with membrane-bound BCL-X L, which seems to be B the active form that inhibits BCL-X L. Act,ve /\ A $112 $136 cytosol BAD Kinase(s) membrane ( Phosphatase(s) !!~ i~i~84184 !~ ~ ~i~iiil~ ~ili~ membrane

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6 Two of these molecules, BID and BAD, lack a COOH-terminal I II hydrophobic segment (Fig. 3). These molecules can translocate Ii II Death Signal between cytosol and classic membrane based family partners such Ii Ii as BCL-2 or BCL-X L. Moreover, mutants of BID and BAD that il II fail to bind their partner proteins were functionally inert. Thus, [] BID and BAD can be envisioned as "death ligands" that bind to A.o;. downstream membrane-based BCL-2 full members, "receptors." These characteristics make BID and BAD candidates for an upstream link between the BCL-2 checkpoint and proximal signal transduction. Extraeellular Survival Factors Reset the BCL-2 Checkpoint by Phosphorylation of BAD. Support for the above thesis comes from the recognition that BAD is rapidly phosphorylated on two serine residues in response to a survival factor, IL-3 (58). These serines represent canonical 14-3-3 binding sites. Phosphorylated BAD seems to be the inactive form incapable of binding BCL-X L and sequestered in the cytosol bound to 14-3-3 (Fig. 6). This would release BCL-X L Fig. 7. Schematic model of BAX activation cell death. A death signal activates BAX, resulting in the translocation of a BAX monomer in the cytosol to homodimerized,integral at membrane sites to promote cell survival. Consistent with this mitochondrial membrane BAX. Downstream effects include the activation of Caspases model, substitution of the serine phosphorylation sites eliminated and a program of mitochondrial dysfunction. binding to 14-3-3 and further enhanced the death-promoting activity of BAD. Despite the similarity of the phosphorylation motifs sur- BID and BAD revealed that their amphipathic BH3 c~ helical domain rounding Serll2 and Ser136 of BAD, distinct BAD kinases seem was critical for both binding to the pocket of antagonists BCL-2 and responsible for the differential phosphorylation of each site after BCL-X L and their death agonist activity (52, 54). These molecules engagement of a single receptor (58, 59). This illustrates the com- provide further support for BH3 as the critical and minimal death plexity of the posttranslational modifications that will regulate this domain. pathway.

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_ Cell Death Si_gn_a_l ~

Pro-apoptotic Death Antic - - O y Checkpoint ~(ced-9) Fig. 8. Schematic model of mammalian cell death pathway. A major checkpoint in the common portion of the pathway is the ratio Caspase (ced-3) of proapoptotic (BAX) to antiapoptotic (BCL-2) members. Cyt C > Apaf,1 (ced-4) < PTP A ~ m ROS q, Death Substrates (PARP) /

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BAX Activation Induces Mitochondrial Dysfunction and Apo- recent identification of egl-1, a BH3-only gene in C. elegans (70), ptosis. Although the ratio of BCL-2:BAX determines the response indicates that the evolving set of BH3-only proteins are a conserved to an apoptotic signal, a question remained as to whether BAX component of the central apoptotic pathway. Cloning of C. elegans ced-3 itself could initiate death and, if so, how. Transient transfection revealed homology to the mammalian enzyme ICE (71). There are more and inducible expression systems for BAX or BAK proved suffi- than 10 ICE-like proteases, now termed caspases (cysteine proteases that cient to induce apoptosis without an additional stimulus (60, 61). cleave after aspartic acid; Ref. (72). Caspases use a cysteinyl residue as BAX-induced death activated caspases which cleaved endogenous the active nucleophile and recognize a cleavage site with an aspartic acid substrates in the nucleus (PARP) and cytosol (D4-GDI; Fig. 7). residue at the P1 position of multiple death substrates. A single ced-4 However, although caspase inhibitors successfully blocked prote- homologue, Apaf-l, which interacts with cytochrome C when released ase activity and could prevent a FAS-induced death, it did not from mitochondria to activate caspases, has also been identified (73). block BAX-induced death. The localization of BCL-2 (15), and Thus, the basic tenets of the cell death pathway seem to be markedly subsequently other family members, to the mitochondria prompted conserved, likely common to all multicellular organisms. Moreover, an assessment of mitochondrial function. Although the cleavage of DNA viruses choose to interfere at these same critical steps to prevent the substrates and the final degradation of DNA was prevented, mito- apoptotic death of their host cells (Fig. 8). Thus, BCL-2, which was chondrial dysfunction still occurred. BAX-induced alterations in provided by follicular lymphoma, like so many genes that are responsible mitochondrial membrane potential, production of reactive oxygen for a human disease, revealed a major genetic and biochemical pathway; species, cytoplasmic vacuolation, and plasma membrane perme- in this case, one that regulates cell survival. The BCL-2 family genes in ability apparently occurred despite the inhibition of measurable this pathway have proven critical for the successful crafting of multicel- caspase activity (60). lular lineages during development, as well as the maintenance of home- BAX has been shown to have both cytosolic and membrane- ostasis in the adult. associated locations. Death signals such as IL-3 withdrawal, stau- rosporine, dexamethasone, and ?/-irradiation induce the transloca- References tion of BAX protein from cytosol to mitochondrial membrane (62, 63). Although BAX forms both homo- and heterodimers, questions 1. Tsujimoto, Y., Gorham, J., Cossman, J., Jaffe, E., and Croce, C. M. The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ remained concerning its native conformation in vivo and which joining. Science (Washington DC), 229: 1390-1393, 1985. moiety was functionally active. Physiological death stimuli result 2. B',tkhshi, A., Jensen, J. P., Goldman, P., Wright, J. J., McBride, O. W., Epstein, A. L., in the translocation of monomeric BAX from the cytosol to the and Korsmeyer, S. J. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around Jn on chromosome 14 and near a transcriptional unit on mitochondria, where it can be cross-linked as a BAX homodimer 18. Cell, 41: 889-906, 1985. (62-64). In contrast, cells protected by BCL-2 demonstrate a block 3. Cleary, M. L., and Sklar, J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a in this process in that BAX does not redistribute or homodimerize transcriptionally active locus on chromosome 18. Proc. Natl. Acad. Sci. USA, 82: in response to a death signal, Moreover, enforced dimerization of 7439-7443, 1985. FKBP-BAX by the bivalent ligand FK1012 resulted in its translo- 4. Cleary, M. L., Smith, S. D., mad Sklar, J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) cation to mitochondria and induced apoptosis (63). Enforced translocation. Cell, 47: 19-28, 1986. dimerization of BAX overrode the protection of BCL-X L and IL-3 5. Seto, M., Jaeger, U., Hockett, R. D., Graninger, W., Bennett, S., Goldman, P., and to kill cells. 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1700s

Stanley J. Korsmeyer

Cancer Res 1999;59:1693s-1700s.

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