Sandra S. Zinkel

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to celldeath (apoptosis), mediating the release of cytochromec from mitochondria in response to signals from ‘death’ receptors known as TNFR1/Fas on the cell surface. It is a member of the pro-apoptotic Bcl-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFα. (fibroblasts). In these Bid−/− cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.

BCL2 family in DNA damage and cell cycle control

Individual BCL2 family members couple apoptosis regulation and cell cycle control in unique ways. Antiapoptotic BCL2 and BCL-xL are antiproliferative by facilitating G0. BAX is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the multi-domain BCL2 family members (MCL-1) and suggest that survival is maintained at the expense of proliferation. The role of BH3-only molecules in cell cycle is more variable. BAD antagonizes both the cell cycle and antiapoptotic functions of BCL2 and BCL-xL through BH3 binding. BID has biochemically separable functions in apoptosis and S-phase checkpoint, determined by post-translational modification. p53-induced PUMA is known only to have apoptotic function. Inhibition of apoptosis is oncogenic, whereas promotion of cell cycle arrest is tumor suppressive. Paradoxically, selected BCL2 family members can be both oncogenic and tumor suppressive. Which of the dual functions predominates is lineage specific and context dependent.

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid−/− neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid−/− mice (−67% and −41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.

A role for proapoptotic BID in the DNA-damage response.

The BCL-2 family of apoptotic proteins encompasses key regulators proximal to irreversible cell damage. The BH3-only members of this family act as sentinels, interconnecting specific death signals to the core apoptotic pathway. Our previous data demonstrated a role for BH3-only BID in maintaining myeloid homeostasis and suppressing leukemogenesis. In the absence of Bid, mice accumulate chromosomal aberrations and develop a fatal myeloproliferative disorder resembling chronic myelomonocytic leukemia. Here, we describe a role for BID in preserving genomic integrity that places BID at an early point in the path to determine the fate of a cell. We show that BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage distinct from its proapoptotic function. We further demonstrate that this role is mediated through BID phosphorylation by the DNA-damage kinase ATM. These results establish a link between proapoptotic Bid and the DNA-damage response.

Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy‐related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL‐CBP expression and to assess the immediate effects of induced MLL‐CBP expression on hematopoiesis. Within days of activating MLL‐CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self‐renewal/proliferation. MLL‐CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL‐CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal γ‐irradiation or N‐ethyl‐N‐nitrosourea (ENU), MLL‐CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy‐induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL‐CBP therapy‐related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.

Catabolite activator protein-induced DNA bending in transcription initiation☆

We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter. Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion. We found that the bend is maintained and even increased upon formation of closed and open complexes. Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation. We now suggest a new model, in which DNA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain. Formation of this structure may facilitate open complex formation. We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter.

Function of BID – a Molecule of the bcl-2 Family – in Ischemic Cell Death in the Brain

Mitochondrial mechanisms, particularly the release of cytochrome c, play a role in the death of nerve and glial cells in cerebral ischemia. We have currently investigated whether BID, a proapoptotic molecule of the bcl-2 family and promoter of the release of cytochrome c is expressed in the brain, activated by cerebral ischemia in vivo, and contributes to ischemic cell death. We found BID in the cytosol of mouse brain and of primary cultured mouse neurons and showed that neuronal BID is a substrate for caspase 8. BID was cleaved in vivo 4 h after transitory occlusion of the middle cerebral artery. Further, BID–/– mice had a significant attenuation of infarction (–67%) and significantly lower release of cytochrome c (–41%). The findings indicate that the proapoptotic molecule BID may contribute to the demise of nerve cells from cerebral ischemia by release of cytochrome c and activation of caspase.

Bid regulates the pathogenesis of neurotropic reovirus.

Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence.

Bid Plays a Role in the DNA Damage Response

A Matters Arising recently published in Cell by Kaufmann et al. (2007) concludes that the BH3-only Bid protein is dispensable for the DNA damage response. Based on our earlier findings that ATM-mediated Bid phosphorylation is essential for S phase arrest and for the regulation of apoptosis in response to DNA damage (Kamer et al., 2005; Zinkel et al., 2005), we would like to emphasize that we reached the opposite conclusion. Based on a careful comparison between the three studies (see below), we believe that the conclusion that Bid is dispensable for the DNA damage response is not supported by all of the available data.

Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress.

Proapoptotic BH3 interacting domain death agonist (Bid), a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage.1, 2, 3 In this study, we demonstrate that Bid functions at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells show an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased checkpoint kinase 1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.