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Lymphocyte survival during immune responses is controlled by the relative expression of pro-and anti-apoptotic molecules, regulating the magnitude, quality, and duration of the response. We investigated the consequences of deleting genes encoding the anti-apoptotic molecules Mcl1 and Bcl2l1 (Bd-[X.sub.L]) from B cells using an inducible system synchronized with expression of activation-induced cytidine deaminase (Aicda) after immunization. This revealed Met1 and not Bcl2l1 to be indispensable for the formation and persistence of germinal centers (GCs). Limiting Mall expression reduced the magnitude of the GC response with an equivalent, but not greater, effect on memory B cell formation and no effect on persistence. Our results identify Mcl1 as the main anti-apoptotic regulator of activated B ceil survival and suggest distinct mechanisms controlling survival of GC and memory B cells. 10.1126/science.1191793

Although Fas-induced apoptosis is thought to require extensive aggregation of pre-assembled Fas trimers (12-15), it has not been resolved whether mFasL, sFasL or both cause cell killing when expressed physiologically [...], Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses (1-3) and prevention of autoimmunity (4,5). Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice (6,7) and humans (8,9). FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding (10,11). Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized nonapoptotic activities. Mice lacking sFasL ([FasL.sup.Δs/Δs]) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL ([FasL.sup.Δm/Δm]) could not kill cells through Fas activation. [FasL.sup.Δm/Δm] mice developed lymphadenopathy and hyper-gamma-globulinaemia, similar to [FasL.sup.gld/gld] mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, [FasL.sup.Δm/Δm] mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in [FasL.sup.gld/gld] mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through nonapoptotic activities.

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development (1-4). Distinct cell [...]

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.

Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-[2.sup.-/-] mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with EIA and Ras oncogenes, caspase-[2.sup.-I-] MEFs grew significantly faster than caspase-[2.sup.+/+] MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse E[mu]-Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-[2.sup.-/-] mice. The caspase-[2.sup.-/-] cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-[2.sup.-/-] MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following y-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein. cell survival | tumorigenesis |cell cycle | proliferation | DNA damage

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemo-resistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bims2A, which is highly selective for Mcl-1. Unlike Noxa, Bims2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bims2A promotes cell killing only when Bcl-[x.sub.L] is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.

Dysfunction of mitochondrial complex I is associated with a wide spectrum of neurodegenerative disorders, including Parkinson's disease (PD). In rodents, inhibition of complex I leads to degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNpc), as seen in PD, through activation of mitochondria-dependent apoptotic molecular pathways. In this scenario, complex I blockade increases the soluble pool of cytochrome c in the mitochondrial intermembrane space through oxidative mechanisms, whereas activation of pro-cell death protein Bax is actually necessary to trigger neuronal death by permeabilizing the outer mitochondrial membrane and releasing cytochrome c into the cytosol. Activation of Bax after complex I inhibition relies on its transcriptional induction and translocation to the mitochondria. How complex I deficiency leads to Bax activation is currently unknown. Using gene-targeted mice, we show that the tumor suppressor p53 mediates Bax transcriptional induction after PD-related complex I blockade in vivo, but it does not participate in Bax mitochondrial translocation in this model, either by a transcription-independent mechanism or through the induction of BH3-only proteins Puma or Noxa. Instead, Bax mitochondrial translocation in this model relies mainly on the JNK-dependent activation of the BH3-only protein Bim. Targeting either Bax transcriptional induction or Bax mitochondrial translocation results in a marked attenuation of SNpc dopaminergic cell death caused by complex I inhibition. These results provide further insight into the pathogenesis of PD neurodegeneration and identify molecular targets of potential therapeutic significance for this disabling neurological illness. apoptosis | Bax | Bim | complexl | 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine

Cell killing is a critical pharmacological activity of imatinib to eradicate Bcr/[Abl.sup.+] leukemias. We found that imatinib kills Bcr/ [Abl.sup.+] leukemic cells by triggering the Bcl-2-regulated apoptotic pathway. Imatinib activated several proapoptotic BH3-only proteins: bim and bmf transcription was increased, and both Bim and Bad were activated posttranslationally. Studies using RNAi and cells from gene-targeted mice revealed that Bim plays a major role in imatinib-induced apoptosis of Bcr/[Abl.sup.+] leukemic cells and that the combined loss of Bim and Bad abrogates this killing. Loss of Bmf or Puma had no effect. Resistance to imatinib caused by Bcl-2 overexpression or loss of Bim (plus Bad) could be overcome by cotreatment with the BH3 mimetic ABT-737. These results demonstrate that Bim and Bad account for most, perhaps all, imatinib-induced killing of Bcr/[Abl.sup.+] leukemic cells and suggest previously undescribed drug combination strategies for cancer therapy. apoptosis | imatinib mesylate | Bcl-2 | cancer | leukemia

T cell receptor/CD3 ligation induces apoptosis in semimature CD[4.sup.+][8.sup.-]HS[A.sup.+] thymocytes, and this helps establish immunological tolerance and constitutes one of the safeguards against autoimmune disease. We analyzed several knockout and transgenic mouse lines and found that T cell receptor/CD3-ligation-induced killing of semimature thymocytes occurred independently of Fas and 'death receptor' signaling in general but required the proapoptotic BH3-only protein Bim and could be inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the Bcl-2 family protein family, provided only minor protection, indicating that the 'apoptosome' functions as an amplifier rather than as an essential initiator of this death program. These results reveal the mechanisms of apoptosis in negative selection of semimature thymocytes and have implications for immunological tolerance and autoimmunity. Bcl-2 family | Fas (APO-1/CD95) | FADD/Mort1 | Apaf-1 | caspase-9

Impaired apoptosis is now recognized to be central to tumor development. Bcl2, activated by chromosomal translocation in human follicular lymphoma, promotes oncogenesis by inhibiting apoptosis. Bim, a distant proapoptotic relative, is emerging as a major physiologic antagonist of Bcl2. Here, we show that loss of Bim is oncogenic. Bim protein levels were elevated in the apoptosis-prone B lymphoid cells of E[micro]-Myc-transgenic mice, and Bimmutant E[micro]-Myc mice had increased numbers of IgM-bearing B cells. E[micro]-Myc-expressing B lymphoid cells deficient in Bim were refractory to apoptosis induced in vitro by cytokine deprivation or antigen receptor cross-linking. Thus, Bim is induced by Myc in B cells and mediates apoptosis. Remarkably, inactivation of even a single allele of Bim accelerated Myc-induced development of tumors, particularly acute B cell leukemia. None of the primary tumors from [Bim.sup.+/-] E[micro]-Myc mice displayed loss of the second allele of Bim. These findings indicate that Bim is a tumor suppressor, at least in B lymphocytes, and is haploinsufficient. Whereas the p19Arf/p53 pathway is frequently mutated in tumors arising in [Bim.sup.+/+] E[micro]-Myc mice, it was unaffected in most Bim-deficient tumors, indicating that Bim reduction is an effective alternative to loss of p53 function.

We used mutant Fas-deficient (Ipr) or Bim-deficient mice to investigate the role of the death receptor and Bcl-2-regulated apoptotic pathways in terminating a physiological T cell response to herpes simplex virus infection. In WT and Ipr mice C[D8.sup.+] antigen-specific T cells were deleted after viral clearance. In contrast, the immune response was not terminated in Bim-deficient mice despite viral clearance, and C[D8.sup.+] antigen-specific T cells accumulated in the spleen. Thus, Bim is dispensable for viral clearance but is necessary for the death of activated T cells when immune responses are terminated. These findings have implications for the therapeutic manipulation of immune responses to infections and immunization.

Author(s): Philippe Bouillet [1]; Jared F. Purton [2]; Dale I. Godfrey [2]; Li-Chen Zhang [1]; Leigh Coultas [1]; Hamsa Puthalakath [1]; Marc Pellegrini [1]; Suzanne Cory [1]; Jerry M. Adams [...]

The cloning of full-length Stra8 cDNA and the partial characterization of the corresponding novel protein are described. Using two-dimensional gel analysis and dephosphorylation, it was found that the two stereoisomers of retinoic acid differentially regulate the phosphorylation status of the Stra8 protein, demonstrated to exist in differently phosphorylated forms. Expression of Stra8 during mouse embryogenesis was limited to the male developing gonads, while in adult mice, its expression was restricted to the premeiotic germ cells. Stra8 protein may thus be involved in the premeiotic phase of spermatogenesis.

Segmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factor Krox-20 and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition to Krox-20 and Hox genes, several members of the Eph subfamily of receptor protein tyrosine kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity, Krox-20 appears to act 'upstream' of Hox genes, but the identity of the 'downstream' effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK gene MDK1 which belongs to the Eph receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression of Hoxa-2 and MDK1 in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of the MDK1 in Hoxa-2 null mutant embryos. A selective lack of MDK1 expression in rhombomere 3 of Hoxa-2 mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating that MDK1 lies downstream of Hoxa-2 in the morphogenetic signaling cascade.

Retinoic acid receptors and retinoic X receptors help differentiate F9 embryonal carcinoma cells in vitro. A study of the expression of various additional retinoic acid (RA)-responsive genes in RAR-alpha and RAR-gamma-null F9 cells and their role in cell differentiation reveals that reexpression of RAR-gamma or overexpression of RAR-alpha re-established target-gene activation and the differentiation potential while overexpression of RAR-beta poorly restored differentiation.

An efficient differential subtractive hybridization cloning strategy has been developed to systematically isolate mouse genes whose expression is induced in P19 cells at different times following exposure to retinoic acid (RA). Fifty different cDNA fragments corresponding to RA-induced genes were isolated, of which 40 represent novel genes. Ten of the isolated cDNAs correspond to known genes, of which four have previously been identified as RA-inducible. One of the novel RA-inducible genes (Stra1) has been studied further, and it appears to correspond to a ligand for Cek5, a receptor protein-tyrosine kinase.

Author(s): Vanessa S. Marsden [1]; Liam O'Connor [1, 2]; Lorraine A. O'Reilly [1]; John Silke [1]; Donald Metcalf [1]; Paul G. Ekert [1, 3]; David C. S. Huang [1]; Francesco [...]

Apoptosis is essential for normal development, tissue homeostasis, and immune function; its altered regulation can trigger cancer, autoimmunity, and degenerative disorders (1). Dismantling of the cell is carried out by [...], Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that share only the BH3 domain with this family. Gene targeting in mice revealed important physiological roles for Bim. Lymphoid and myeloid cells accumulated, T cell development was perturbed, and most older mice accumulated plasma cells and succumbed to autoimmune kidney disease. Lymphocytes were refractory to apoptotic stimuli such as cytokine deprivation, calcium ion flux, and microtubule perturbation but not to others. Thus, Bim is required for hematopoietic homeostasis and as a barrier to autoimmunity. Moreover, particular death stimuli appear to activate apoptosis through distinct BH3-only proteins.

Author(s): Philippe Bouillet; Jared F. Purton; Dale I. Godfrey; Li-Chen Zhang; Leigh Coultas; Hamsa Puthalakath; Marc Pellegrini; Suzanne Cory; Jerry M. Adams; Andreas Strasser Nature 415, 922-926 (2002). The number [...]