Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

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

Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics

Background: The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.Methods and Findings: We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.Conclusions: Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy

Lepton Polarization and Forward-Backward Asymmetries in b -> s tau+ tau-

We study the spin polarizations of both tau leptons in the decay b -> s tau+ tau-. In addition to the polarization asymmetries involving a single tau, we construct asymmetries for the case where both polarizations are simultaneously measured. We also study forward-backward asymmetries with polarized tau's. We find that a large number of asymmetries are predicted to be large, >~ 10%. This permits the measurement of all Wilson coefficients and the b-quark mass, thus allowing the standard model (SM) to be exhaustively tested. Furthermore, there are many unique signals for the presence of new physics. For example, asymmetries involving triple-product correlations are predicted to be tiny within the SM, O(10^{-2}). Their observation would be a clear signal of new physics.Comment: 21 pages, LaTeX, 4 figures (included). Paper somewhat reorganized, references greatly expanded, conclusions unchange

Supersymmetric Contributions to Bs->K+K-

Inspired by the existing calculation of B->piK decays in supersymmetry (SUSY), we evaluate the dominant SUSY contributions to Bs->K+K-. We show that the observables of this process can be significantly modified in the presence of SUSY. In particular, the branching ratio can be increased considerably compared to the prediction of the standard model (SM). The effect is even more dramatic for the CP-violating asymmetries A_dir and A_mix. These asymmetries, expected to be small in the SM (A_dir is predicted to take only positive values), change drastically with SUSY contributions. The measurement of these observables can therefore be used to detect the presence of physics beyond the SM, and put constraints on its parameters.Comment: 17 pages, 2 figures. This new version contains one added reference and some minor style change

Safety and Toxicity of Catheter Gene Delivery to the Pulmonary Vasculature in a Patient with Metastatic Melanoma

Overview summary Transcatheter delivery of HLA-B7 DNA and cationic liposomes into a segment of a pulmonary artery was safely performed in 1 patient with tumor nodules in the lung. No immunologic or organ toxicities were observed. Percutaneous catheter gene delivery has been performed in humans. Further refinements of this approach may lead to useful treatments for a variety of human diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63196/1/hum.1994.5.9-1089.pd

Three heavy jet events at hadron colliders as a sensitive probe of the Higgs sector

Assuming that a non-standard neutral Higgs with an enhanced Yukawa coupling to a bottom quark is observed at future hadron experiments, we propose a method for a better understanding of the Higgs sector. Our procedure is based on "counting" the number of events with heavy jets (where "heavy" stands for a c or b jet) versus b jets, in the final state of processes in which the Higgs is produced in association with a single high p_T c or b jet. We show that an observed signal of the type proposed, at either the Tevatron or the LHC, will rule out the popular two Higgs doublet model of type II as well as its supersymmetric version - the Minimal Supersymmetric Standard Model (MSSM), and may provide new evidence in favor of some more exotic multi Higgs scenarios. As an example, we show that in a version of a two Higgs doublet model which naturally accounts for the large mass of the top quark, our signal can be easily detected at the LHC within that framework. We also find that such a signal may be observable at the upgraded Tevatron RunIII, if the neutral Higgs in this model has a mass around 100 GeV and \tan\beta > 50 and if the efficiency for distinguishing a c jet from a light jet will reach the level of 50%.Comment: Revtex, 11 pages, 4 figures embedded in the text. Main changes with respect to Version 1: Numerical results re-calculated using the CTEQ5L pdf, improved discussion on the experimental consequences, new references added. Conclusions remain unchanged. As will appear in Phys. Rev.

A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance 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-xL 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

Prospects for heavy supersymmetric charged Higgs boson searches at hadron colliders

We investigate the production of a heavy charged Higgs boson at hadron colliders within the context of the MSSM. A detailed study is performed for all important production modes and basic background processes for the t\bar{t}b\bar{b} signature. In our analysis we include effects of initial and final state showering, hadronization, and principal detector effects. For the signal production rate we include the leading SUSY quantum effects at high \tan\beta>~ mt/mb. Based on the obtained efficiencies for the signal and background we estimate the discovery and exclusion mass limits of the charged Higgs boson at high values of \tan\beta. At the upgraded Tevatron the discovery of a heavy charged Higgs boson (MH^+ >~ 200 GeV) is impossible for the tree-level cross-section values. However, if QCD and SUSY effects happen to reinforce mutually, there are indeed regions of the MSSM parameter space which could provide 3\sigma evidence and, at best, 5\sigma charged Higgs boson discovery at the Tevatron for masses M_H^+<~ 300 GeV and M_H^+<~ 250 GeV, respectively, even assuming squark and gluino masses in the (500-1000) GeV range. On the other hand, at the LHC one can discover a H^+ as heavy as 1 TeV at the canonical confidence level of 5\sigma; or else exclude its existence at 95% C.L. up to masses ~ 1.5 TeV. Again the presence of SUSY quantum effects can be very important here as they may shift the LHC limits by a few hundred GeV.Comment: Latex2e, 44 pages, 15 figures, 6 tables, uses JHEP3.sty, axodraw.sty. Comments added. Discussion on QCD factors clarified. Added discussion on uncertainties. Change of presentation of Tables 4 and 5 and Fig.6. Results and conclusions unchanged. Version accepted in JHE

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function

Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis

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