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The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1-α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1-α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.

The war on cancer that began some 40 years ago with the discovery of oncogenes is starting to be won. We feel fortunate to have contributed to several advances. Here we recall how molecular biology became our scientific passion, how we met from opposite ends of the earth, and how our 50-year odyssey has taken us from gene expression through immunogenetics to exploring the molecular basis of cancer and cell death. We describe the scientific discoveries that motivated us and remarkable scientists who influenced us. We sketch our studies that clarified the role of chromosome translocations in cancer and established the value of genetically engineered mouse models of tumorigenesis. Finally, we outline how our findings with many talented close colleagues on cell death regulation stimulated the development of remarkable new anticancer agents called BH3 mimetics, which are encouraging hope that many more malignancies will become controllable and even curable.

Drugs targeting various pro-survival BCL-2 family members (''BH3 mimetics'') have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5' untranslated region (5'UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.

It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins’ apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a “Membrane-mediated Permissive” model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.

Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-XL-specific inhibitor A-1331852, or Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which reduced pro-survival signals. We also tested the MCL-1 inhibitor in primary samples from 13 MCL patients, using CD40L-expressing feeder cells to model their microenvironmental support. Notably, all unstimulated primary MCL samples were very sensitive to S63845, but the CD40L stimulation attenuated their sensitivity. Mass cytometric analysis revealed that the stimulation likely conveyed protection by elevating BCL-XL and MCL-1. Accordingly, sensitivity of the CD40L-stimulated cells to S63845 was substantially restored by co-treatment with venetoclax, the BCL-XL-specific inhibitor or ibrutinib. Overall, our findings indicate that MCL-1 is very important for survival of MCL cells and that the MCL-1 inhibitor, both alone and together with ibrutinib, venetoclax or a BCL-XL inhibitor, offers promise for novel improved MCL therapies.

Impaired apoptosis plays a central role in cancer development and limits the efficacy of conventional cytotoxic therapies. Deepening understanding of how opposing factions of the BCL-2 protein family switch on apoptosis and of their structures has driven development of a new class of cancer drugs that targets various pro-survival members by mimicking their natural inhibitors, the BH3-only proteins. These 'BH3 mimetic' drugs seem destined to become powerful new weapons in the arsenal against cancer. Successful clinical trials of venetoclax/ABT-199, a specific inhibitor of BCL-2, have led to its approval for a refractory form of chronic lymphocytic leukaemia and to scores of on-going trials for other malignancies. Furthermore, encouraging preclinical studies of BH3 mimetics that target other BCL-2 pro-survival members, particularly MCL-1, offer promise for cancers resistant to venetoclax. This review sketches the impact of the BCL-2 family on cancer development and therapy, describes how interactions of family members trigger apoptosis and discusses the potential of BH3 mimetic drugs to advance cancer therapy.

The BCL-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide programme that is essential for development, tissue homeostasis and immunity. Too little apoptosis can promote cancer and autoimmune diseases; too much apoptosis can augment ischaemic conditions and drive neurodegeneration. We discuss the biochemical, structural and genetic studies that have clarified how the interplay between members of the BCL-2 family on mitochondria sets the apoptotic threshold. These mechanistic insights into the functions of the BCL-2 family are illuminating the physiological control of apoptosis, the pathological consequences of its dysregulation and the promising search for novel cancer therapies that target the BCL-2 family.

Developing potent molecules that inhibit Bcl-2 family mediated apoptosis affords opportunities to treat cancers via reactivation of the cell death machinery. We describe the hit-to-lead development of selective Bcl-XL inhibitors originating from a high-throughput screening campaign. Small structural changes to the hit compound increased binding affinity more than 300-fold (to IC50 < 20 nM). This molecular series exhibits drug-like characteristics, low molecular weights (Mw < 450), and unprecedented selectivity for Bcl-XL. Surface plasmon resonance experiments afford strong evidence of binding affinity within the hydrophobic groove of Bcl-XL. Biological experiments using engineered Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on Bcl-XL for survival) and Bax/Bak deficient MEFs (insensitive to selective activation of Bcl-2-driven apoptosis) support a mechanism-based induction of apoptosis. This manuscript describes the first series of selective small-molecule inhibitors of Bcl-XL and provides promising leads for the development of efficacious therapeutics against solid tumors and chemoresistant cancer cell lines.

SummaryIn stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2–α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other’s surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

Mutations that impair apoptosis contribute to cancer development and reduce the effectiveness of conventional anti-cancer therapies. These insights and understanding of how the B cell lymphoma (BCL)-2 protein family governs apoptosis have galvanized the search for a new class of cancer drugs that target its pro-survival members by mimicking their natural antagonists, the BCL-2 homology (BH)3-only proteins. Successful initial clinical trials of the BH3 mimetic venetoclax/ABT-199, specific for BCL-2, have led to its recent licensing for refractory chronic lymphocytic leukemia and to multiple ongoing trials for other malignancies. Moreover, preclinical studies herald the potential of emerging BH3 mimetics targeting other BCL-2 pro-survival members, particularly myeloid cell leukemia (MCL)-1, for multiple cancer types. Thus, BH3 mimetics seem destined to become powerful new weapons in the arsenal against cancer. This review sketches the discovery of the BCL-2 family and its impact on cancer development and therapy; describes how interactions of family members trigger apoptosis; outlines the development of BH3 mimetic drugs; and discusses their potential to advance cancer therapy.

As many oncogenic changes, such as Myc overexpression, promote apoptosis, the survival of emerging neoplastic clones may often initially depend upon endogenous levels of particular pro-survival members of the Bcl-2 protein family. Pertinently, we recently showed that in lymphoma-prone Eμ-myc transgenic mice, which overexpress Myc in all B-lymphoid cells, endogenous Bcl-x(L) is critical for the survival, as well as the expansion of preneoplastic B-lymphoid cells and the development of malignant disease. This discovery raised the possibility that pharmacological blockade of Bcl-x(L) might impede Myc-driven lymphoma development. Indeed, we report here that treatment of preleukaemic Eμ-myc transgenic mice with the Bcl-2 homology (BH)3 mimetic drug ABT-737, which inhibits Bcl-x(L), as well as Bcl-2 and Bcl-w, augmented apoptosis of preneoplastic B-lymphoid cells, reduced their numbers and greatly prolonged lymphoma-free survival. These findings reveal that BH3 mimetic drugs may provide a prophylactic strategy to prevent the development of certain tumours, particularly those driven by deregulated Myc expression. Moreover, such treatment may help in the management of patients with hereditary cancer syndromes and perhaps also in the prevention of tumour relapses.

Apoptosis, the major form of programmed cell death in metazoan organisms, plays critical roles in normal development, tissue homeostasis and immunity, and its disturbed regulation contributes to many pathological states, including cancer, autoimmunity, infection and degenerative disorders. In vertebrates, it can be triggered either by engagement of 'death receptors' of the tumour necrosis factor receptor family on the cell surface or by diverse intracellular signals that act upon the Bcl-2 protein family, which controls the integrity of the mitochondrial outer membrane through the complex interactions of family members. Both pathways lead to cellular demolition by dedicated proteases termed caspases. This review discusses the groundbreaking experiments from many laboratories that have clarified cell death regulation and galvanised efforts to translate this knowledge into novel therapeutic strategies for the treatment of malignant and perhaps certain autoimmune and infectious diseases.

VEGF deprivation induces Bim expression in tumor endothelial cells, and Bim is needed for anti-VEGF–driven endothelial cell death and tumor shrinkage., For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature.

Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in γ-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from γ-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

Evasion of apoptosis is critical for tumorigenesis, and sustained survival of nascent neoplastic cells may depend upon the endogenous levels of pro-survival BCL-2 family members. Indeed, previous studies using gene-targeted mice revealed that BCL-XL, but surprisingly not BCL-2, is critical for the development of c-MYC-induced pre-B/B lymphomas. However, it remains unclear whether another pro-survival BCL-2 relative contributes to their development. MCL-1 is an intriguing candidate, because it is required for cell survival during early B-lymphocyte differentiation. It is expressed abnormally high in several types of human B-cell lymphomas and is implicated in their resistance to chemotherapy. To test the B-cell intrinsic requirement for endogenous MCL-1 in lymphoma development, we conditionally deleted Mcl-1 in B-lymphoid cells of Eμ-Myc transgenic mice. We found that MCL-1 loss in early B-lymphoid progenitors delayed MYC-driven lymphomagenesis. Moreover, the lymphomas that arose when MCL-1 levels were diminished appeared to have been selected for reduced levels of BIM and/or increased levels of BCL-XL. These results underscore the importance of MCL-1 in lymphoma development and show that alterations in the levels of other cell death regulators can compensate for deficiencies in MCL-1 expression.

Apoptosis is essential for tissue homeostasis, particularly in the hematopoietic compartment, where its impairment can elicit neoplastic or autoimmune diseases. Whether stressed cells live or die is largely determined by interplay between opposing members of the Bcl-2 protein family. Bcl-2 and its closest homologs promote cell survival, but two other factions promote apoptosis. The BH3-only proteins sense and relay stress signals, but commitment to apoptosis requires Bax or Bak. The BH3-only proteins appear to activate Bax and Bak indirectly, by engaging and neutralizing their pro-survival relatives, which otherwise constrain Bax and Bak from permeabilizing mitochondria. The Bcl-2 family may also regulate autophagy and mitochondrial fission/fusion. Its pro-survival members are attractive therapeutic targets in cancer and perhaps autoimmunity and viral infections.

The pro-apoptotic BH3-only members of the Bcl2 family, crucial initiators of cell death, are activated by a diverse array of developmental cues or experimentally applied stress stimuli. We have investigated, through gene targeting in mice, the biological roles for the BH3-only family member HRK (also known as DP5) in apoptosis regulation. Hrk gene expression was found to be restricted to cells and tissues of the central and peripheral nervous systems. Sensory neurons from mice lacking Hrk were less sensitive to apoptosis induced by nerve growth factor (NGF) withdrawal, consistent with the induction of Hrk following NGF deprivation. By contrast, cerebellar granule neurons that upregulate Hrk upon transfer to low-K+ medium underwent apoptosis normally under these conditions in the absence of Hrk. Furthermore, loss of Hrk was not sufficient to rescue the neuronal degeneration in lurcher mutant mice. Despite previous reports, no evidence was found for Hrk expression or induction in growth-factor-dependent haematopoietic cell lines following withdrawal of their requisite cytokine, and haematopoietic progenitors lacking HRK died normally in response to cytokine deprivation. These results demonstrate that HRK contributes to apoptosis signalling elicited by trophic factor withdrawal in certain neuronal populations but is dispensable for apoptosis of haematopoietic cells.

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)–only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-xl, Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-xl–like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-xl and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.

A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2–like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.

Numerous p53 target genes have been implicated in DNA damage–induced apoptosis signaling, but proapoptotic Bcl-2 (B-cell leukemia 2) family members of the BH3 (Bcl-2 homolog region [BH] 3)–only subgroup appear to play the critical initiating role. In various types of cultured cells, 3 BH3-only proteins, namely Puma (p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting mediator of cell death), have been shown to initiate p53-dependent as well as p53-independent apoptosis in response to DNA damage and treatment with anticancer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids. To assess the in vivo relevance of these findings, we subjected mice lacking Puma, Noxa, or Bim to whole-body γ-radiation or the glucocorticoid dexamethasone and compared lymphocyte survival with that in wild-type and BCL2–transgenic mice. Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of lymphocytes from the effects of γ-radiation in vivo, and loss of Bim provided lower but significant protection in most lymphocytes, whereas Noxa deficiency had no impact. Furthermore, both Puma and Bim were found to contribute significantly to glucocorticoid-induced killing. Our results thus establish that Puma and Bim are key initiators of γ-radiation– and glucocorticoid-induced apoptosis in lymphoid cells in vivo.

BH3-only members of the Bcl-2 intracellular protein family, which include Bim, Bmf, Bik, Bad, Bid, Puma, Noxa and Hrk, mediate many developmentally programmed and induced cytotoxic signals. They have key roles in development, tissue homeostasis, immunity and tumor suppression, and compounds mimicking them are promising anti-cancer agents. Their activity is normally constrained by transcriptional and/or diverse post-transcriptional controls. When activated, these death ligands engage pro-survival Bcl-2-like proteins via the BH3 domain, inactivating their function. Bim and Puma bind all the pro-survival proteins, whereas others, such as Noxa and Bad, engage distinct subsets and exhibit complementary killing. Hence, multiple pro-survival proteins must be inactivated to unleash Bax and Bak, which drive apoptosis. Whether certain BH3-only proteins also directly activate Bax/Bak remains controversial.

The BH3-only proteins of the Bcl-2 family initiate apoptosis through the activation of Bax-like relatives. Loss of individual BH3-only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik−/−bim−/− and bim−/− mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik−/−bim−/− males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik−/−bim−/− males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development.

How the Bcl-2 family of proteins regulate programmed cell death triggered by developmental cues and in response to multiple stress signals is of intense interest (Adams 2003; Danial and Korsmeyer 2004). Whereas cell survival is promoted by Bcl-2 itself and several close relatives (Bcl-xL, Bcl-w, Mcl-1, and A1), which bear three or four conserved Bcl-2 homology (BH) regions, apoptosis is driven by two other subfamilies. The initial signal for cell death is conveyed by the diverse group of BH3-only proteins, including Bad, Bid, Bim, Puma, and Noxa, which have in common only the small BH3 interaction domain (Huang and Strasser 2000). However, Bax or Bak (multidomain proteins containing BH1-BH3) are required for commitment to cell death (Lindsten et al. 2000; Cheng et al. 2001; Wei et al. 2001; Zong et al. 2001). When activated, they can permeabilize the outer membrane of mitochondria and release proapoptogenic factors (e.g., cytochrome c) needed to activate the caspases that dismantle the cell (Adams 2003; Danial and Korsmeyer 2004; Green and Kroemer 2004). Interactions between members of these three factions of the Bcl-2 family dictate whether a cell lives or dies. When BH3-only proteins have been activated, for example, in response to DNA damage, they can bind via their BH3 domain to a groove on their prosurvival relatives (Sattler et al. 1997). How the BH3-only and Bcl-2-like proteins control the activation of Bax and Bak, however, remains poorly understood (Adams 2003; Danial and Korsmeyer 2004). Most attention has focused on Bax. This soluble monomeric protein (Hsu et al. 1997; Wolter et al. 1997) normally has its membrane-targeting domain inserted into its groove, probably accounting for its cytosolic localization (Suzuki et al. 2000; Schinzel et al. 2004). Several unrelated peptides/proteins have been proposed to modulate Bax activity (for review, see Lucken-Ardjomande and Martinou 2005), but their physiological relevance remains to be established. Alternatively, Bax may be activated via direct engagement by certain BH3-only proteins, the best documented being the active truncated form of Bid, tBid (Wei et al. 2000; Kuwana et al. 2002; Roucou et al. 2002). As discussed elsewhere (Adams 2003), the oldest model, in which Bcl-2 directly engages Bax (Oltvai et al. 1993), has become problematic because Bcl-2 is membrane bound while Bax is cytosolic, and their interaction seems highly dependent on certain detergents used for cell lysis (Hsu and Youle 1997). Nevertheless, it is well established that the BH3 region of Bax can mediate association with Bcl-2 (Zha and Reed 1997; Wang et al. 1998) and that Bcl-2 prevents the oligomerization of Bax, even though no heterodimers can be detected (Mikhailov et al. 2001). Thus, whether the prosurvival proteins restrain Bax activation directly or indirectly remains uncertain (see Discussion). Although Bax and Bak seem in most circumstances to be functionally equivalent (Lindsten et al. 2000; Wei et al. 2001), substantial differences in their regulation would be expected from their distinct localization in healthy cells. Unlike Bax, which is largely cytosolic, Bak resides in complexes on the outer membrane of mitochondria and on the endoplasmic reticulum of healthy cells (Wei et al. 2000; Zong et al. 2003). Nevertheless, on receipt of cytotoxic signals, both Bax and Bak change conformation, and Bax translocates to the organellar membranes, where both Bax and Bak then form homo-oligomers that can associate, leading to membrane permeabilization (Hsu et al. 1997; Wolter et al. 1997; Antonsson et al. 2001; Wei et al. 2001; Mikhailov et al. 2003). Since Bak, unlike Bax, is normally located at its site of action, how is it kept in check to prevent inappropriate cell death? We were prompted to investigate Bak regulation by recent evidence that it can form complexes with Mcl-1 (Cuconati et al. 2003) and that Mcl-1 is degraded at an early stage of apoptosis (Cuconati et al. 2003; Nijhawan et al. 2003). Here we report evidence from binding and functional studies that Bak is subject to negative regulation specifically by Mcl-1 and Bcl-xL but not other prosurvival family members. Thus, contrary to expectation, the prototypic guardian Bcl-2 is unable to prevent Bak activation. We show that stimuli from DNA damage drive BH3-only proteins to displace Bak from Mcl-1 and Bcl-xL, allowing Bak to self-associate and trigger apoptosis. We also report that the association of Noxa with Mcl-1 can trigger Mcl-1 degradation. Our demonstration that a subset of prosurvival family members controls Bak may explain the varied phenotypes observed on disruption of the prosurvival genes (Ranger et al. 2001) and has important implications for current efforts to develop drugs that regulate apoptosis by targeting the Bcl-2 family (Cory et al. 2003).

Previously, we showed that the proteasome inhibitor bortezomib/Velcade (formerly PS-341) synergizes with the protein tumor necrosis factor α–related apoptosis-inducing ligand (TRAIL), a ligand for certain death receptors, to induce apoptosis in cell lines derived from prostate and colon cancers. Because apoptosis is often triggered by BH3-only proteins of the Bcl-2 family, we have explored the hypothesis that bortezomib contributes to the apoptosis by up-regulating their levels. Indeed, bortezomib induced increases of Bik and/or Bim in multiple cell lines but not notably of two other BH3-only proteins (Puma and Bid) nor other family members (Bax, Bak, Bcl-2, and Bcl-xL). The increase in Bik levels seems to reflect inhibition by bortezomib of its proteasome-mediated degradation. Importantly, both Bik and Bim seem central to the proapoptotic function of bortezomib, because mouse embryo fibroblasts in which the genes for both Bik and Bim had been disrupted were refractory to its cytotoxic action. Similarly, the synergy between bortezomib and TRAIL in killing human prostate cancer cells was impaired in cells in which both Bik and Bim were down-regulated by RNA interference. Further evidence that bortezomib acts through the mitochondrial pathway regulated by the Bcl-2 family is that deficiency for APAF-1, which acts downstream of Bcl-2, also blocked its apoptotic effect. These results implicate BH3-only proteins, in particular both Bik and Bim, as important mediators of the antitumor action of bortezomib and establish their role in its enhancement of TRAIL-induced apoptosis.

On December 15, 2014, the hematology and leukemia research communities lost one of their greatest leaders to pancreatic cancer. Through a remarkable 60-year research career, Donald Metcalf led the discovery and characterization of the regulators of blood cell production. The legacy of his work is not only that blood cells have become the best-understood complex biological system, but also that the clinical applications of its regulators have already benefited over 20 million cancer patients.

Activation of the caspases that initiate apoptosis typically requires cognate scaffold proteins, including CED-4 in Caenorhabditis elegans, Apaf-1 in mammals and Dark in Drosophila. Each scaffold protein oligomerizes procaspases into a complex called the apoptosome, but the regulation and biological roles of the scaffolds differ. Whereas CED-4 is restrained by the Bcl-2 homologue CED-9, Apaf-1 is inhibited by its WD40 repeat region, until it is activated by cytochrome c, derived from damaged mitochondria. Although Dark also has a WD40 region, its activation does not seem to involve cytochrome c. CED-4 is essential for apoptosis in the worm and Dark for many apoptotic responses in the fly, but the Apaf-1/caspase-9 system probably amplifies rather than initiates the mammalian caspase cascade.

Pro-apoptotic BH3-only Bcl-2 family members in vertebrate model organisms suitable for genetic experimentation

Tissue homeostasis is regulated by apoptosis, the cell-suicide programme that is executed by proteases called caspases. The Bcl2 family of intracellular proteins is the central regulator of caspase activation, and its opposing factions of anti- and pro-apoptotic members arbitrate the life-or-death decision. Apoptosis is often impaired in cancer and can limit conventional therapy. A better understanding of how the Bcl2 family controls caspase activation should result in new, more effective therapeutic approaches.

During lymphocyte development, the assembly of genes coding for antigen receptors occurs by the combinatorial linking of gene segments. The stochastic nature of this process gives rise to lymphocytes that can recognize self-antigens, thereby having the potential to induce autoimmune disease. Such autoreactive lymphocytes can be silenced by developmental arrest or unresponsiveness (anergy), or can be deleted from the repertoire by cell death. In the thymus, developing T lymphocytes (thymocytes) bearing a T-cell receptor (TCR)-CD3 complex that engages self-antigens are induced to undergo programmed cell death (apoptosis), but the mechanisms ensuring this 'negative selection' are unclear. We now report that thymocytes lacking the pro-apoptotic Bcl-2 family member Bim (also known as Bcl2l11) are refractory to apoptosis induced by TCR-CD3 stimulation. Moreover, in transgenic mice expressing autoreactive TCRs that provoke widespread deletion, Bim deficiency severely impaired thymocyte killing. TCR ligation upregulated Bim expression and promoted interaction of Bim with Bcl-XL, inhibiting its survival function. These findings identify Bim as an essential initiator of apoptosis in thymocyte-negative selection.

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.

Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5' to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases.

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease–activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2–like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2–like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

Objective: The objective of this study was to investigate the renal effects of celecoxib, a cyclooxygenase-2 (COX-2) specific nonsteroidal anti-inflammatory drug (NSAID). Subjects and treatment: Six volume-depleted, conscious, chronically instrumented dogs received four treatments using a cross-over design, with at least 13 days between treatments. An i.v. bolus dose (3, 10, and 30 μmol/kg) of vehicle, indomethacin, celecoxib, or 6-MNA, the active metabolite of nabumetone, were administered with an hour between doses. Methods: Renal function was assessed at baseline and 30 min after each dose. Results: Treatment with indomethacin or celecoxib resulted in a dose-dependent reduction in renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow, sodium excretion, and fractional sodium excretion. GFR was reduced by 27% with 30 μmol/kg indomethacin (p < 0.05) and by 58% with 30 μmol/kg celecoxib (p < 0.5). Similarly, RPF was significantly (p < 0.05) reduced by 33% and 65% with 30 μmol/kg indomethacin and celecoxib, respectively. Reductions in renal function with celecoxib occurred at a therapeutic index (determined by comparison anti-edemic activities of the drugs in rat paw carrageenan assays) equal to or lower than indomethacin. In contrast, treatment with vehicle or 6-MNA resulted in no reductions in renal function. Conclusion: Administration of either celecoxib (specific COX-2 inhibitor) or indomethacin (selective COX-1 inhibitor) resulted in significant reductions in renal function. COX-2 specific inhibitors do not appear to lack the renal effects of NSAIDs.

Damage to DNA produces cell cycle arrest, apoptosis, or both. The response in cells with p53 tumor suppressor function involves transcriptional changes, but whether that holds for cells lacking active p53, as in most tumors, is not known. Better characterization of the DNA damage response in tumors lacking p53 function is relevant to cytotoxic therapy. We have explored whether gamma-irradiated p53-null mouse T lymphoma cells undergo marked changes in transcription. Their arrest in G2/M prior to apoptosis required transcription. Transcripts whose abundance altered on irradiation were sought by subtractive hybridization, and 1010 candidate clones from two oppositely enriched cDNA populations were sequenced. Hybridization revealed small (3-fold) increases or decreases in the transcripts of more than 15 genes, including some implicated in cell cycle control (e.g., BTG, Bap1) or apoptosis (e.g., STAT1, calpain), but no marked changes like those associated with other forms of T-cell death. Moreover, the expression of some critical apoptosis regulators, such as Bcl-2 family members, did not change. Hence, the G2/M arrest and apoptosis in the irradiated p53-null lymphoma appears to involve modest expression changes for many genes, but post-transcriptional alterations may be more critical.

The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2–controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.

The pathways to lymphoid neoplasia have been explored in a number of transgenic models. Because B lymphoid malignancies often involve translocation of an oncogene (e.g. myc, bcl-2, cyclin D1) to an immunoglobulin locus, resulting in its deregulated expression, the consequences of oncogene overexpression in lymphocytes can be evaluated with transgenes driven by an immunoglobulin regulatory element, such as an enhancer from the IgH locus. Mice bearing such transgenes have provided insight into the preneoplastic state, including alterations in the control of cellular proliferation, differentiation or apoptosis. They have also allowed studies on oncogene cooperation in vivo and the modulating effect of genetic background. Briefly reviewed here are the models studied in the authors' laboratories. Mice bearing myc and bcl-2 transgenes have received most attention but others studied include abl, ras, cyclin D1 and bmi-1 oncogenes. Also discussed is a new transgenic vector that should facilitate transgenic approaches to non-lymphoid leukemias. The vector bears elements from the promoter region of the vav gene, which is expressed almost exclusively in hematopoietic cells. It has proven capable of driving transgene expression throughout the hematopoietic compartment, including progenitor cells and their precursors. This novel vector should aid studies on many aspects of hematopoiesis, including the modeling of leukemogenesis.

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, thein vivoassociation studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 could be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain was necessary and sufficient for their interaction. Apaf-1 did not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-xL, Bcl-w, A1, Mcl-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein–Barr virus BHRF-1. Endogenous Apaf-1 also failed to coimmunoprecipitate with endogenous Bcl-2 or Bcl-xL, or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli did not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly.

Transduction of hematopoietic stem cells with a novel retrovirus has allowed long-term expression of human Bcl-2 in multiple hematopoietic lineages. Thy-1.2 lo Sca-1 + H-2K hi stem cells enriched from the bone marrow of 5-fluorouracil–treated (Ly5-2) mice were infected with the bcl -2 retrovirus and injected into (Ly5-1) irradiated recipients. Analysis at 5 months indicated that reconstitution of hematopoiesis occurred predominantly from donor-derived (Ly5-2 + ) stem cells and that, in half the mice (18 of 35), most blood cells derived from virally transduced stem cells. The level of Bcl-2 expression achieved with the retroviral vector approached that of a well-characterized transgenic vector and could be sustained for life in several blood cell lineages. In the 25 mice assessed at 10 months, human Bcl-2 was readily detectable in 62 ± 22% of Ly5-2 + peripheral blood leukocytes. More detailed analysis of a cohort killed between 14 and 20 months established that human Bcl-2 protein could be detected in B and T lymphocytes, granulocytes, macrophages, and some immature erythroid cells. Furthermore, hematopoietic stem cells from the bone marrow of these mice maintained Bcl-2 expression in hematopoietic tissues of secondary recipients for at least another 19 months. These data provide clear evidence for efficient infection of primitive hematopoietic stem cells and for maintenance of proviral expression for over 2.5 years, the lifespan of mice. The level of exogenous Bcl-2 was sufficient to enhance survival of B and T lymphoid cells, granulocytes, and myeloid colony-forming cells cultured under suboptimal conditions, but hematopoiesis in the mice was not notably perturbed.

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolatedbcl-wgene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated thebcl-wgene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. Thebcl-w−/−mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

The vav gene is expressed in all hematopoietic but few other cell types. To explore its unusual compartment-wide regulation, we cloned the murine gene, sequenced its promoter region, identified DNase I hypersensitive (HS) sites in the chromatin, and tested their promoter activity with a β-galactosidase (β-gal) reporter gene in cell lines and transgenic mice. Whereas fibroblasts had no HS sites, a myeloid and an erythroid cell line contained five, located 0.2 kb (HS1), 1.9 kb (HS2), and 3.6 kb (HS3) upstream from the transcription start and 0.6 kb (HS4) and 10 kb (HS5) downstream. A vav DNA fragment including HS1 promoted β-gal expression in a myeloid but not a fibroblast line. Expression in leukocytes of transgenic mice also required HS2 and HS5. Only hematopoietic organs contained β-gal, but virtually all β-gal+ cells were B or T lymphocytes. Expression was always variegated (mosaic), and the proportion of β-gal+ cells declined with lymphoid maturation and animal age. Thus, these vav regulatory elements promoted hematopoietic-specific expression in vivo, at least in lymphocytes, but the transgene was sporadically silenced. Maintaining pan-hematopoietic expression may require additional vavelements or an alternative reporter.