Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65.

PubMed

Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Domínguez-Rodríguez, Jorge Ramiro; Jave-Suárez, Luis F; De Célis-Carrillo, Ruth; Aguilar-Lemarroy, Adriana; Gómez-Lomeli, Paulina; Ortiz-Lazareno, Pablo Cesar

2013-02-28

In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm antileukemic potential.

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization.

PubMed

Shah, O Jameel; Lin, Xiaoyu; Li, Leiming; Huang, Xiaoli; Li, Junling; Anderson, Mark G; Tang, Hua; Rodriguez, Luis E; Warder, Scott E; McLoughlin, Shaun; Chen, Jun; Palma, Joann; Glaser, Keith B; Donawho, Cherrie K; Fesik, Stephen W; Shen, Yu

2010-07-13

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This "addiction" can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization

PubMed Central

Shah, O. Jameel; Lin, Xiaoyu; Li, Leiming; Huang, Xiaoli; Li, Junling; Anderson, Mark G.; Tang, Hua; Rodriguez, Luis E.; Warder, Scott E.; McLoughlin, Shaun; Chen, Jun; Palma, Joann; Glaser, Keith B.; Donawho, Cherrie K.; Fesik, Stephen W.; Shen, Yu

2010-01-01

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility. PMID:20616035

Contribution of either YY1 or BclXL-induced inhibition by the NO-donor DETANONOate in the reversal of drug resistance, both in vitro and in vivo. YY1 and BclXL are overexpressed in prostate cancer.

PubMed

Huerta-Yepez, Sara; Baritaki, Stavroula; Baay-Guzman, Guillermina; Hernandez-Luna, Marco A; Hernandez-Cueto, Angeles; Vega, Mario I; Bonavida, Benjamin

2013-02-28

Nitric oxide (NO) donors have been shown to activate or inhibit constitutively-activated survival/anti-apoptotic pathways, such as NF-κB, in cancer cells. We report here that treatment of drug-resistant human prostate carcinoma cell lines with high levels (500-1000 μM) of the NO-donor DETANONOate sensitized the resistant tumor cells to apoptosis by CDDP and the combination was synergistic. We hypothesized that DETANONOate inhibits previously identified NF-κB-regulated resistant factors such as Yin Yang 1 (YY1) and Bcl-2/BclXL. Lysates from tumor cells treated with DETANONOate showed inhibition of YY1 and BclXL expressions. Transfection with either YY1 or BclXL siRNA resulted in the inhibition of both YY1 and BclXL expressions and sensitized the cells to CDDP apoptosis. Mice bearing PC-3 tumor xenografts and treated with the combination of DETANONOate and CDDP resulted in significant inhibition of tumor growth; treatment with single agent alone did not have any effect on tumor growth. Analysis of patients TMA tissues with prostatic cancer revealed higher expression of both YY1 and BclXL as a function of tumor grades and their levels were directly correlated. Thus, both YY1 and BclXL are potential prognostic biomarkers. Overall, the above findings suggest that one mechanism of DETANONOate-induced sensitization of resistant tumor cells to CDDP correlated with the inhibition of NF-κB and its targets YY1 and BclXL. The examination of the combination of NO donors and cytotoxic therapy in the treatment of resistant prostate cancer may be warranted. Published by Elsevier Inc.

BAX/BCL-XL gene expression ratio inversely correlates with disease progression in chronic myeloid leukemia.

PubMed

Gonzalez, Mariana S; De Brasi, Carlos D; Bianchini, Michele; Gargallo, Patricia; Moiraghi, Beatriz; Bengió, Raquel; Larripa, Irene B

2010-10-15

BCR-ABL fusion gene is implicated in the pathogenesis of chronic myeloid leukemia (CML), encoding the oncoprotein p210(BCR-ABL) with anti-apoptotic activity. The inability to undergo apoptosis is an important mechanism of drug resistance and neoplastic evolution in CML. The gene transcript expression of mitochondrial apoptotic related genes BAX and BCL-XL was evaluated by quantitative Real Time PCR (qPCR) in vitro in K562 cells and in vivo in peripheral blood of 66 CML patients in different stages of the disease: 13 cases at diagnosis, 34 in chronic phase (CP), 10 in accelerated phase (AP) and 9 in blast crisis (BC). Our results in K562 cells showed that all treatments with different tyrosine kinase inhibitors (TKIs) induced a decreased expression of the antiapoptotic oncogene BCL-XL, whereas the proapoptotic gene BAX remains constant with minor modifications. A significantly lower BAX/BCL-XL expression ratio (mean±SEM) than a group of healthy individuals (4.8±0.59) were observed in CML patients at diagnosis (1.28 ± 0.16), in AP (1.14±0.20), in BC (1.16±0.30) and in 18% of cases of patients in CP (2.71±0.40). Most CP cases (82%) showed a significantly increased ratio (10.03±1.30), indicating that the treatment with TKIs efficiently inhibited the expression of BCL-XL by blocking BCR-ABL oncoprotein. The BAX/BCL-XL ratio showed a significant inverse correlation (Spearman P<0.0001) with BCR-ABL/ABL relative expression indicating that low BAX/BCL-XL was associated with disease progression. Accordingly, the follow up of a cohort of eight cases during 6months from diagnosis showed that while the BAX/BCL-XL ratio rapidly increased after treatment in seven cases with good evolution, it decreased in the single case that showed rapid evolution and short survival. Our data suggest that BAX/BCL-XL expression ratio may be a sensitive monitor of disease progression and an early predictor of TKI therapy responsiveness in CML patients. Copyright © 2010 Elsevier Inc. All

Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Guodong; Peng, Tao; Zhou, Xuhong

2013-11-01

Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messengermore » RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of

[Behavior in the forced-swimming test and expression of BDNF and Bcl-xl genes in the rat brain].

PubMed

Berezova, I V; Shishkina, G T; Kalinina, T S; Dygalo, N N

2011-01-01

A single exposure of rats to the forced-swimming stress decreased BDNF mRNA levels in the cortex and increased Bcl-xl gene expression in the hippocampus and amygdala 24 h after the stress. The animals demonstrated a depressive-like behavior and elevated blood corticosterone level. There was a significant negative correlation between BDNF mRNA level in the cortex and immobility time during swimming. Repeated exposure to swimming stress caused the elevation of the hippocampal BDNF mRNA level assessed 24 h after the second swimming session. The data suggest that stress-induced down-regulation of cortical BDNF gene expression and behavioral despair in the forced-swimming test may be interrelated. The increase in the BDNF and Bcl-xl mRNA levels may contribute to the mechanisms protecting the brain against negative effects of stress.

Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray.

PubMed

Zha, Hongbin; Raffeld, Mark; Charboneau, Lu; Pittaluga, Stefania; Kwak, Larry W; Petricoin, Emanuel; Liotta, Lance A; Jaffe, Elaine S

2004-02-01

Overexpression of Bcl-2 protein has been known to play a role in the pathogenesis of follicular lymphoma (FL). However, 10-15% of FLs are negative for Bcl-2 by immunohistochemistry, raising the possibility that another gene product(s) may provide prosurvival signal(s). We used reverse phase protein microarray to analyze lysates of follicle center cells isolated by laser capture microdissection from: Bcl-2+ FL, Bcl-2- FL and reactive follicular hyperplasia (FH) (nine cases each group). TUNEL assay confirmed similar and reduced levels of apoptosis in Bcl-2+ FL and Bcl-2- FL, indicating the likelihood of Bcl-2-independent inhibition of apoptosis. Arrays were quantitatively analyzed with antibodies to proteins involved in the apoptotic pathway. As expected, Bcl-2 levels were up to eight-fold higher in Bcl-2+ FL than in FH and Bcl-2- FL. However, there was no difference in levels of Mcl-1 and survivin among these three groups. Bcl-X(L) showed a trend for increased expression in Bcl-2- FL as compared with Bcl-2+ FL, although the differences did not reach statistical significance (P>0.1). The increase in Bcl-X(L) may provide an alternative antiapoptotic signal in FL negative for Bcl-2 protein. Interestingly, Bax expression was higher in FL (Bcl-2+ or -) than in FH (P=0.001). Notably, phospho-Akt (Ser-473) was increased in FL (Bcl-2+ or -) (P<0.03) with increased phospho-Bad (Ser-136), as compared with levels in FH. The activation of the Akt/Bad pathway provides further evidence of prosurvival signals in FL, independent of Bcl-2 alone. These data suggest that nodal FL represents a single disease with a final common biochemical pathway.

Anti-apoptotic BCL-2 family proteins in acute neural injury

PubMed Central

Anilkumar, Ujval; Prehn, Jochen H. M.

2014-01-01

Cells under stress activate cell survival and cell death signaling pathways. Cell death signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (BCL-2) proteins. In this review, we summarize current knowledge on the control of neuronal survival, development and injury by anti-apoptotic BCL-2 family proteins. We discuss overlapping and differential effects of the individual family members BCL-2, BCL-extra long (BCL-XL), myeloid cell leukemia 1 (MCL-1), and BCL2-like 2 (BCL-W) in the control of survival during development and pathophysiological processes such as trophic factor withdrawal, ischemic injury, excitotoxicity, oxidative stress and energy stress. Finally we discuss recent evidence that several anti-apoptotic BCL-2 proteins influence mitochondrial bioenergetics and control neuronal Ca2+ homeostasis independent of their classical role in cell death signaling. PMID:25324720

Anti-apoptotic BCL-2 family proteins in acute neural injury.

PubMed

Anilkumar, Ujval; Prehn, Jochen H M

2014-01-01

Cells under stress activate cell survival and cell death signaling pathways. Cell death signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (BCL-2) proteins. In this review, we summarize current knowledge on the control of neuronal survival, development and injury by anti-apoptotic BCL-2 family proteins. We discuss overlapping and differential effects of the individual family members BCL-2, BCL-extra long (BCL-XL), myeloid cell leukemia 1 (MCL-1), and BCL2-like 2 (BCL-W) in the control of survival during development and pathophysiological processes such as trophic factor withdrawal, ischemic injury, excitotoxicity, oxidative stress and energy stress. Finally we discuss recent evidence that several anti-apoptotic BCL-2 proteins influence mitochondrial bioenergetics and control neuronal Ca(2+) homeostasis independent of their classical role in cell death signaling.

Advanced glycation end products influence oral cancer cell survival via Bcl-xl and Nrf-2 regulation in vitro.

PubMed

Ko, Shun-Yao; Ko, Hshin-An; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chen, Yi-Ting; Yu, Ya-Hui; Yang, Shu-Han; Chang, Shu-Shing

2017-05-01

An irreversible non-enzymatic reaction between carbohydrates and proteins results in the formation of advanced glycation end products (AGEs). AGEs have been demonstrated to be a risk factor of complications in patients with diabetes mellitus (DM). Previous studies have suggested that patients with DM exhibit a higher rate of metastasis of oral cancer and a lower cancer-associated survival rate. The receptor for AGEs (RAGE) has been associated with angiogenesis and an increase in cancer malignancy. Previous studies have suggested that AGE-RAGE regulates cell migration via extracellular signal-regulated kinase (ERK) phosphorylation. Nuclear factor-erythroid 2-related factor 2 (Nrf-2) is associated with the regulation of tumor protein p53 (p53) and the apoptotic response of oral cancer cells. AGEs are associated with oral cancer; however, the mechanism underlying this association remains to be elucidated. The present study hypothesized that AGEs regulate Nrf-2 and downstream pathways through ERK phosphorylation. The results of the current study demonstrated that AGEs inhibit the expression of Nrf-2, p53 and Bcl-2 associated × apoptosis regulator, and increase the expression of apoptosis regulator Bcl-x protein. The effect of AGEs was inhibited through the use of the PD98059. The present study demonstrated that AGEs regulate the downstream pathways Nrf-2 and Bcl-xl via ERK phosphorylation. It is suggested that AGEs regulate the survival of oral cancer cells via Nrf-2 and Bcl-xl through p53 regulation, which explains the poor prognosis of patients with DM who have oral cancer.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

PubMed

Shishkina, Galina T; Kalinina, Tatyana S; Bulygina, Veta V; Lanshakov, Dmitry A; Babluk, Ekaterina V; Dygalo, Nikolay N

2015-01-01

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids

PubMed Central

Kalinina, Tatyana S.; Bulygina, Veta V.; Lanshakov, Dmitry A.; Babluk, Ekaterina V.

2015-01-01

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons. PMID:26624017

rno-miR-665 targets BCL2L1 (Bcl-xl) and increases vulnerability to propofol in developing astrocytes.

PubMed

Sun, Wen-Chong; Pei, Ling

2016-07-01

Propofol exerts a cytotoxic influence over immature neurocytes. Our previous study revealed that clinically relevant doses of propofol accelerated apoptosis of primary cultured astrocytes of developing rodent brains via rno-miR-665 regulation. However, the role of rno-miR-665 during the growth spurt of neonatal rodent brains in vivo is still uncertain. Post-natal day 7 (P7) rats received a single injection of propofol 30 mg/kg intraperitoneally (i.p.), and neuroapoptosis of hippocampal astrocytes was analyzed by immunofluorescence and scanning electron microscopy. The differential expression of rno-miR-665, BCL2L1 (Bcl-xl), and cleaved caspase 3 (CC3) was surveyed by qRT-PCR and western blotting. In addition, the utility of A-1155463, a highly potent and BCL2L1-selective antagonist, was aimed to assess the contribution of BCL2L1 for neuroglial survival. Following the intraventricular injection of lentivirus rno-miR-665, neuroprotection was detected by 5-point scale measurement. The single dose of propofol 30 mg/kg triggered dose-dependent apoptosis of developing hippocampal astrocytes. Meanwhile, propofol triggered both rno-miR-665 and CC3, and depressed BCL2L1, which was predicted as one target gene of rno-miR-665. Combination treatment with A-1155463 and propofol induced lower mRNA and protein levels of BCL2L1 and more CC3 activation than propofol treatment alone in vivo. The lentivirus-mediated knockdown of rno-miR-665 elevated BCL2L1 and attenuated CC3 levels, whereas up-regulation of rno-miR-665 suppressed BCL2L1 and induced CC3 expression in vivo. More importantly, rno-miR-665 antagomir infusion improved neurological outcomes of pups receiving propofol during the brain growth spurt. Rno-miR-665, providing a potential target for alternative therapeutics for pediatric anesthesia, is susceptible to propofol by negatively targeting antiapoptotic BCL2L1. Relatively little is known about the association between exposure of astrocytes to brief propofol

Upregulation of microRNA-876 Induces Endothelial Cell Apoptosis by Suppressing Bcl-Xl in Development of Atherosclerosis.

PubMed

Xu, Kaicheng; Liu, Peng; Zhao, Yue

2017-01-01

The injury and apoptotic cell death of endothelial cells hallmark the development of atherosclerosis (AS), characterized by dysregulation of lipid homeostasis, immune responses, and formation of coronary plaques. However, the mechanisms underlying the initiation of endothelial cell apoptosis remain ill-defined. Recent evidence suggests a role of microRNAs in the processes of AS-associated endothelial cell apoptosis. Thus, we studied this question in the current study. AS was developed in ApoE (-/-) mice suppled with high-fat diet (HFD), compared to ApoE (-/-) mice suppled with normal diet (ND). Mouse endothelial cells were isolated from the aortic arch using flow cytometry based on their expression of Pecam-1. Oxidized low-density lipoprotein (ox-LDL) were used to treat human aortic endothelial cells (HAECs) as an in vitro model for AS. Gene expression was quantified by RT-qPCR and protein levels were analyzed by Western blotting. Apoptosis was evaluated by FITC Annexin V Apoptosis essay and by TUNEL staining. Prediction of the binding between miRNAs and 3'-UTR of mRNA from the target gene was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. HFD mice, but not ND mice, developed AS in 12 weeks. Significantly reduced endothelial cell marks and significantly increased mesenchymal cell marks were detected in the aortic arch of the HFD mice, compared to the ND mice. The endothelial cell apoptosis was significantly higher in HFD mice, seemingly due to functional suppression of protein translation of anti-apoptotic Bcl-Xl protein through upregulation of miR-876. Similar results were obtained from in vitro study. Inhibition of miR-876 abolished the effects of ox-LDL-induced apoptotic cell death of HAECs. AS-associated endothelial cell apoptosis may partially result from downregulation of Bcl-Xl, through upregulation of miR-876 that binds and suppresses translation of Bcl-Xl mRNA. © 2017 The Author(s). Published by S. Karger AG, Basel.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

PubMed

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues.

PubMed

Ohta, K; Iwai, K; Kasahara, Y; Taniguchi, N; Krajewski, S; Reed, J C; Miyawaki, T

1995-11-01

The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm.

PubMed

Cayli, Sevil; Sakkas, Denny; Vigue, Lynne; Demir, Ramazan; Huszar, Gabor

2004-05-01

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction, <4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (>80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

PubMed

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-12-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

PubMed Central

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-01-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

CXCR4 Chemokine Receptor Signaling Induces Apoptosis in Acute Myeloid Leukemia Cells via Regulation of the Bcl-2 Family Members Bcl-XL, Noxa, and Bak*

PubMed Central

Kremer, Kimberly N.; Peterson, Kevin L.; Schneider, Paula A.; Meng, X. Wei; Dai, Haiming; Hess, Allan D.; Smith, B. Douglas; Rodriguez-Ramirez, Christie; Karp, Judith E.; Kaufmann, Scott H.; Hedin, Karen E.

2013-01-01

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted. PMID:23798675

An antiapoptotic Bcl-2 family protein index predicts the response of leukaemic cells to the pan-Bcl-2 inhibitor S1

PubMed Central

Zhang, Z; Liu, Y; Song, T; Xue, Z; Shen, X; Liang, F; Zhao, Y; Li, Z; Sheng, H

2013-01-01

Background: Bcl-2-like members have been found to be inherently overexpressed in many types of haematologic malignancies. The small-molecule S1 is a BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1 and Bcl-XL. Methods: The lethal dose 50 (LD50) values of S1 in five leukaemic cell lines and 41 newly diagnosed leukaemia samples were tested. The levels of Bcl-2 family members and phosphorylated Bcl-2 were semiquantitatively measured by western blotting. The interactions between Bcl-2 family members were tested by co-immunoprecipitation. The correlation between the LD50 and expression levels of Bcl-2 family members, alone or in combination, was analysed. Results: S1 exhibited variable sensitivity with LD50 values ranging >2 logs in both established and primary leukaemic cells. The ratio of pBcl-2/(Bcl-2+Mcl-1) could predict the S1 response. Furthermore, we demonstrated that pBcl-2 antagonised S1 by sequestering the Bak and Bim proteins that were released from Mcl-1, andpBcl-2/Bak, pBcl-2/Bax and pBcl-2/Bim complexes cannot be disrupted by S1. Conclusion: A predictive index was obtained for the novel BH3 mimetic S1. The shift of proapoptotic proteins from being complexed with Mcl-1 to being complexed with pBcl-2 was revealed for the first time, which is the mechanism underlying the index value described herein. PMID:23558901

The expression of bcl-2 and bcl-6 protein in normal and malignant transitional epithelium.

PubMed

Lin, Zhenhua; Kim, Hankyeom; Park, Hongseok; Kim, Youngsik; Cheon, Jun; Kim, Insun

2003-08-01

The bcl-2 proto-oncogene plays a key role in cell longevity by preventing apoptosis. Bcl-2 is important in developing and maintaining the normal function of lymphoid and epithelial tissues. The bcl-6 protein is a 96 kDa nuclear protein selectively expressed in mature B cells within normal germinal centers as well as in their transformed counterparts in diffuse large B cell lymphoma. Recently, the bcl-6 protein has also been reported to be expressed in normal skin and epidermal neoplasms. In this study, 47 cases of transitional cell carcinomas (TCCs) were immunohistochemically studied for bcl-2 and bcl-6 protein expression. The results showed that bcl-2 was expressed only on basal layer cells, whereas bcl-6 expression was restricted to the superficial layers in the normal transitional epithelium. Von Brunn's nests showed strong immunostaining to bcl-2, but were negative to bcl-6. Among 47 TCCs, 15 (32.6%) and 29 (61.7%) cases were positive for bcl-2 and bcl-6, respectively. Compared with the normal transitional epithelium, the expression of bcl-2 was significantly decreased, whereas bcl-6 expression was significantly increased in TCCs. Additionally, the strong expression of bcl-6 had a positive correlation with the histopathologic grade of TCC. In conclusion, bcl-2 and bcl-6 proteins may play a role in the pathogenesis of TCCs, and bcl-6 expression reflects histopathologic grade.

mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

PubMed

Faber, Anthony C; Coffee, Erin M; Costa, Carlotta; Dastur, Anahita; Ebi, Hiromichi; Hata, Aaron N; Yeo, Alan T; Edelman, Elena J; Song, Youngchul; Tam, Ah Ting; Boisvert, Jessica L; Milano, Randy J; Roper, Jatin; Kodack, David P; Jain, Rakesh K; Corcoran, Ryan B; Rivera, Miguel N; Ramaswamy, Sridhar; Hung, Kenneth E; Benes, Cyril H; Engelman, Jeffrey A

2014-01-01

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl

PubMed Central

Wang, Guanyu; Sharma, Sherven; Dong, Qinghua

2012-01-01

Background The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice. Methodology and Results The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers. Conclusion and Significance These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer. PMID:23240061

Bag3 promotes resistance to apoptosis through Bcl-2 family members in non-small cell lung cancer.

PubMed

Zhang, Yong; Wang, Jian-Hua; Lu, Qiang; Wang, Yun-Jie

2012-01-01

In non-small cell lung cancer (NSCLC) certain molecular characteristics, which are related to molecular alterations have been investigated. These are responsible for both the initiation and maintenance of the malignancy in lung cancer. The aim of this study was to evaluate the influence of Bag3 (Bcl-2 associated athanogene 3) in the regulation of apoptosis on NSCLC. Bag3 and Hsp70 expression were examined by immunohistochemistry to confirm their potential roles in the prevalence of NSCLC. We also established human normal bronchial epithelial cells and HOP-62 cell line as the model to analyze cell apoptosis and the expression of Hsp70, Bcl-XL and Bcl-2, which were affected by Bag3. In this study, we found that Bag3 and Hsp70 are highly expressed in few tissues and cell lines of NSCLC. Bag3 inhibits apoptosis in human normal bronchial epithelial cell lines and sustain the survival of NSCLC cells. Bag3, Hsp70, Bcl-XL and Bcl-2 are up-regulated in NSCLC cell lines. At the same time, the silencing of Bag3 results in diminishing protein levels of Bcl-XL and Bcl-2. The results of immunoprecipitation identified that Bag3 could interact with Hsp70, Bcl-XL and Bcl-2 NSCLC cells directly or indirectly. We conclude that NSCLC cells were protected from apoptosis through increasing Bag3 expression and consequently promoted the expression of Bcl-XL and Bcl-2.

mTOR Inhibition Specifically Sensitizes Colorectal Cancers with KRAS or BRAF Mutations to BCL-2/BCL-XL Inhibition by Suppressing MCL-1

PubMed Central

Faber, Anthony C.; Coffee, Erin M.; Costa, Carlotta; Dastur, Anahita; Ebi, Hiromichi; Hata, Aaron N.; Yeo, Alan T.; Edelman, Elena J.; Song, Youngchul; Tam, Ah Ting; Boisvert, Jessica L.; Milano, Randy J.; Roper, Jatin; Kodack, David P.; Jain, Rakesh K.; Corcoran, Ryan B.; Rivera, Miguel N.; Ramaswamy, Sridhar; Hung, Kenneth E.; Benes, Cyril H.; Engelman, Jeffrey A.

2014-01-01

Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. PMID:24163374

Phenylbutyrate Attenuates the Expression of Bcl-XL, DNA-PK, Caveolin-1, and VEGF in Prostate Cancer Cells1

PubMed Central

Goh, Meidee; Chen, Feng; Paulsen, Michelle T; Yeager, Ann M; Dyer, Erica S; Ljungman, Mats

2001-01-01

Abstract Phenylbutyrate (PB) is a histone deacetylase inhibitor that has been shown to induce differentiation and apoptosis in various cancer cell lines. Although these effects are most likely due to modulation of gene expression, the specific genes and gene products responsible for the effects of PB are not well characterized. In this study, we used cDNA expression arrays and Western blot to assess the effect that PB has on the expression of various cancer and apoptosis-regulatory gene products. We show that PB attenuates the expression of the apoptosis antagonist Bcl-XL, the double-strand break repair protein DNA-dependent protein kinase, the prostate progression marker caveolin -1, and the pro-angiogenic vascular endothelial growth factor. Furthermore, PB was found to act in synergy with ionizing radiation to induce apoptosis in prostate cancer cells. Taken together, our results point to the possibility that PB may be an effective anti-prostate cancer agent when used in combination with radiation or chemotherapy and for the inhibition of cancer progression. PMID:11571633

XIAP impairs mitochondrial function during apoptosis by regulating the Bcl-2 family in renal cell carcinoma.

PubMed

Chen, Chao; Liu, Tian Shu; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-05-01

Efficient apoptosis requires Bcl-2 family-mediated mitochondrial outer membrane permeabilization (MOMP), which releases pro-apoptotic proteins to the cytosol, activating apoptosis and inhibiting X-linked inhibitor of apoptosis protein (XIAP). XIAP is a member of the inhibitors of apoptosis protein family whose expression is elevated in many cancer types and participates in the release of pro-apoptotic proteins. To explore the association between XIAP and the Bcl-2 family, and the influence of XIAP on mitochondria, RNA interference of XIAP was performed in Caki-1 cells and the dynamic change in the levels of related proteins was compared with the original Caki-1 cells upon induction of apoptosis. Upon knockdown of XIAP, the release of cytochrome c (Cyt-c), second mitochondria-derived activator of caspase (Smac) and apoptotic protease activating factor 1 (Apaf-1) from mitochondria proceeded normally, whereas in Caki-1 cells, the release of these pro-apoptotic proteins was significantly prolonged, and incomplete. Downregulation of XIAP through small interfering RNA resulted in an increase of apoptosis and a marked decrease in Bcl-2 and Bcl-xl levels at 3 h. Additionally, the regulation of the level of XIAP protein affected the specific ratios of Bcl-2/Bax and Bcl-xl/Bax, which play decisive roles in cell death. In the present study, it was revealed that XIAP can feed back to mitochondria, delaying Cyt-c and Apaf-1 release. Furthermore, XIAP can limit the release of its inhibitor Smac with the involvement of Bcl-2 family proteins.

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

PubMed

Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

2015-11-26

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199).

PubMed

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-11-13

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2(High)) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2(High) cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2(High) NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2(Low) NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2(Low)) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

Bcl-2 protein expression in mucoepidermoid carcinoma of salivary glands: a single institution experience.

PubMed

Janjua, Omer Sefvan; Qureshi, Sana Mehmood; Khan, Tariq Sarfraz; Alamgir, Wajiha

2012-01-01

Mucoepidermoid carcinoma is the most common salivary gland tumor with varying behavior among different histopathological grades. The objective of this study was to determine the expression of Bcl-2 protein in mucoepidermoid carcinoma (MEC) and to correlate with histological grades. The records of 40 cases of MEC were collected from the histopathology department. Fresh slides were prepared and fresh diagnoses were made using the grading criteria for MEC. Immunohistochemical markers for Bcl-2 were applied and the results analyzed using the chi-square test. Of 40 cases, 20 were males and 20 were females. The range in age of the patients was 6 to 67 years mean (SD) was 42.6 (1.85) years. Twenty-two were low grade (55%), 11 high grade (27.5%) and 7 (17.5%) were intermediate grade MEC. Among these 40 cases, Bcl-2 expression was positive in 24 cases and negative in 16 cases. In 22 cases of low-grade MEC, 19 were positive while only 3 were negative. In high-grade tumors, all 11 cases were found to have a negative expression of Bcl-2 protein. In intermediate-grade MEC, 5 cases showed positive expression while only 2 cases showed negative expression. Bcl-2 protein expression showed positive expression in low-grade and negative expression in high-grade MEC. Intermediate grade showed more than 50% positive results for Bcl-2. Correlation between grades of MEC and expression of Bcl-2 is statistically significant and can be used for the depicting the prognosis of MEC along with other prognostic and clinico-pathological parameters.

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)

PubMed Central

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-01-01

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2High) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2Low) that could benefit from BCL-XL (navitoclax)-driven combination therapy. PMID:26565405

Targeting MUC1-C suppresses BCL2A1 in triple-negative breast cancer.

PubMed

Hiraki, Masayuki; Maeda, Takahiro; Mehrotra, Neha; Jin, Caining; Alam, Maroof; Bouillez, Audrey; Hata, Tsuyoshi; Tagde, Ashujit; Keating, Amy; Kharbanda, Surender; Singh, Harpal; Kufe, Donald

2018-01-01

B-cell lymphoma 2-related protein A1 (BCL2A1) is a member of the BCL-2 family of anti-apoptotic proteins that confers resistance to treatment with anti-cancer drugs; however, there are presently no agents that target BCL2A1. The MUC1-C oncoprotein is aberrantly expressed in triple-negative breast cancer (TNBC) cells, induces the epithelial-mesenchymal transition (EMT) and promotes anti-cancer drug resistance. The present study demonstrates that targeting MUC1-C genetically and pharmacologically in TNBC cells results in the downregulation of BCL2A1 expression. The results show that MUC1-C activates the BCL2A1 gene by an NF-κB p65-mediated mechanism, linking this pathway with the induction of EMT. The MCL-1 anti-apoptotic protein is also of importance for the survival of TNBC cells and is an attractive target for drug development. We found that inhibiting MCL-1 with the highly specific MS1 peptide results in the activation of the MUC1-C→NF-κB→BCL2A1 pathway. In addition, selection of TNBC cells for resistance to ABT-737, which inhibits BCL-2, BCL-xL and BCL-W but not MCL-1 or BCL2A1, is associated with the upregulation of MUC1-C and BCL2A1 expression. Targeting MUC1-C in ABT-737-resistant TNBC cells suppresses BCL2A1 and induces death, which is of potential therapeutic importance. These findings indicate that MUC1-C is a target for the treatment of TNBCs unresponsive to agents that inhibit anti-apoptotic members of the BCL-2 family.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

PubMed

Leverson, Joel D; Phillips, Darren C; Mitten, Michael J; Boghaert, Erwin R; Diaz, Dolores; Tahir, Stephen K; Belmont, Lisa D; Nimmer, Paul; Xiao, Yu; Ma, Xiaoju Max; Lowes, Kym N; Kovar, Peter; Chen, Jun; Jin, Sha; Smith, Morey; Xue, John; Zhang, Haichao; Oleksijew, Anatol; Magoc, Terrance J; Vaidya, Kedar S; Albert, Daniel H; Tarrant, Jacqueline M; La, Nghi; Wang, Le; Tao, Zhi-Fu; Wendt, Michael D; Sampath, Deepak; Rosenberg, Saul H; Tse, Chris; Huang, David C S; Fairbrother, Wayne J; Elmore, Steven W; Souers, Andrew J

2015-03-18

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics. Copyright © 2015, American Association for the Advancement of Science.

Synthesis and Biological Evaluation of Apogossypolone Derivatives as Pan-active Inhibitors of Anti-apoptotic B-Cell Lymphoma/Leukemia-2 (Bcl-2) Family Proteins

PubMed Central

Wei, Jun; Kitada, Shinichi; Stebbins, John L.; Placzek, William; Zhai, Dayong; Wu, Bainan; Rega, Michele F.; Zhang, Ziming; Cellitti, Jason; Yang, Li; Dahl, Russell; Reed, John C.; Pellecchia, Maurizio

2010-01-01

Overexpression of anti-apoptotic Bcl-2 family proteins is commonly related with tumor maintenance, progression, and chemoresistance. Inhibition of these anti-apoptotic proteins is an attractive approach for cancer therapy. Guided by nuclear magnetic resonance (NMR) binding assays, a series of 5, 5′ substituted compound 6a (Apogossypolone) derivatives was synthesized and identified pan-active antagonists of anti-apoptotic Bcl-2 family proteins, with binding potency in the low micromolar to nanomolar range. Compound 6f inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2 and Mcl-1 with IC50 values of 3.10, 3.12 and 2.05 μM, respectively. In a cellular assay, 6f potently inhibits cell growth in several human cancer cell lines in a dose-dependent manner. Compound 6f further displays in vivo efficacy in transgenic mice and demonstrated superior single-agent antitumor efficacy in a PPC-1 mouse xenograft model. Together with its negligible toxicity, compound 6f represents a promising drug lead for the development of novel apoptosis-based therapies for cancer. PMID:21033669

Locating herpesvirus Bcl-2 homologs in the specificity landscape of anti-apoptotic Bcl-2 proteins

PubMed Central

Foight, Glenna Wink; Keating, Amy E.

2015-01-01

Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2 and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2, but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9 nM for binding to KSBcl-2 and >1000-fold specificity over human Bcl-2 proteins, and a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners. PMID:26009469

Apogossypol Derivatives as Pan-active Inhibitors of Anti-apoptotic B-cell lymphoma/leukemia-2 (Bcl-2) Family Proteins

PubMed Central

Wei, Jun; Kitada, Shinichi; Rega, Michele F.; Stebbins, John L.; Zhai, Dayong; Cellitti, Jason; Yuan, Hongbin; Emdadi, Aras; Dahl, Russell; Zhang, Ziming; Yang, Li; Reed, John C.; Pellecchia, Maurizio

2009-01-01

Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a series of 5, 5′ substituted Apogossypol derivatives was synthesized that resulted in potent pan-active inhibitors of anti-apoptotic Bcl-2 family proteins. Compound 8r inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl-1 with IC50 values of 0.76, 0.32, 0.28 and 0.73 μM, respectively. The compound also potently inhibits cell growth of human lung cancer and BP3 human B-cell lymphoma cell lines with EC50 values of 0.33 and 0.66 μM, respectively. Compound 8r shows little cytotoxicity against bax−/−bak−/− cells, indicating that it kills cancers cells via the intented mechanism. The compound also displays in vivo efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Together with its improved chemical, plasma and microsomal stability relative to compound 2 (Apogossypol), compound 8r represents a promising drug lead for the development of novel apoptosis-based therapies for cancer. PMID:19555126

Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer.

PubMed

Merino, D; Lok, S W; Visvader, J E; Lindeman, G J

2016-04-14

The last three decades have seen significant progress in our understanding of the role of the pro-survival protein BCL-2 and its family members in apoptosis and cancer. BCL-2 and other pro-survival family members including Mcl-1 and BCL-XL have been shown to have a key role in keeping pro-apoptotic 'effector' proteins BAK and BAX in check. They also neutralize a group of 'sensor' proteins (such as BIM), which are triggered by cytotoxic stimuli such as chemotherapy. BCL-2 proteins therefore have a central role as guardians against apoptosis, helping cancer cells to evade cell death. More recently, an increasing number of BH3 mimetics, which bind and neutralize BCL-2 and/or its pro-survival relatives, have been developed. The utility of targeting BCL-2 in hematological malignancies has become evident in early-phase studies, with remarkable clinical responses seen in heavily pretreated patients. As BCL-2 is overexpressed in ~75% of breast cancer, there has been growing interest in determining whether this new class of drug could show similar promise in breast cancer. This review summarizes our current understanding of the role of BCL-2 and its family members in mammary gland development and breast cancer, recent progress in the development of new BH3 mimetics as well as their potential for targeting estrogen receptor-positive breast cancer.

BCL-2 family proteins: changing partners in the dance towards death.

PubMed

Kale, Justin; Osterlund, Elizabeth J; Andrews, David W

2018-01-01

The BCL-2 family of proteins controls cell death primarily by direct binding interactions that regulate mitochondrial outer membrane permeabilization (MOMP) leading to the irreversible release of intermembrane space proteins, subsequent caspase activation and apoptosis. The affinities and relative abundance of the BCL-2 family proteins dictate the predominate interactions between anti-apoptotic and pro-apoptotic BCL-2 family proteins that regulate MOMP. We highlight the core mechanisms of BCL-2 family regulation of MOMP with an emphasis on how the interactions between the BCL-2 family proteins govern cell fate. We address the critical importance of both the concentration and affinities of BCL-2 family proteins and show how differences in either can greatly change the outcome. Further, we explain the importance of using full-length BCL-2 family proteins (versus truncated versions or peptides) to parse out the core mechanisms of MOMP regulation by the BCL-2 family. Finally, we discuss how post-translational modifications and differing intracellular localizations alter the mechanisms of apoptosis regulation by BCL-2 family proteins. Successful therapeutic intervention of MOMP regulation in human disease requires an understanding of the factors that mediate the major binding interactions between BCL-2 family proteins in cells.

Type I interferon (IFN-alpha/beta) rescues B-lymphocytes from apoptosis via PI3Kdelta/Akt, Rho-A, NFkappaB and Bcl-2/Bcl(XL).

PubMed

Badr, Gamal; Saad, Heba; Waly, Hanan; Hassan, Khadega; Abdel-Tawab, Hanem; Alhazza, Ibrahim M; Ahmed, Emad A

2010-01-01

Although IFN-alpha was reported to promote the survival of peripheral B-lymphocytes via the PI3-kinase-Akt pathway, the triggered signalling pathways involved in the protection of B cell from apoptosis need to be clarified. Using flow cytometry and western blot analysis, we have found that type 1 IFNs (IFN-alpha/beta) protect human B cells in culture from spontaneous apoptosis and from apoptosis mediated by anti-CD95 agonist, in a dose- and time-dependant manner. IFN-alpha/beta-mediated anti-apoptotic effect on human B cells was totally abrogated by blockade of IFNR1 chain. Our data indicate that PI3Kdelta, Rho-A, NFkappaB and Bcl-2/Bcl(XL) are active downstream of IFN receptors and are the major effectors of IFN-alpha/beta-rescued B cells from apoptosis. Furthermore, immunohistochemical results show marked reduction in numbers of CD20 positive B cell in both spleen and Peyer's patches from mice treated with anti-IFNR1 blocking antibody compared with control group. Moreover, ultrastructural observations of these organs show an obvious increase in apoptotic cells from mice treated with anti-IFNR1 blocking antibody. Our results provide more details about the triggered signalling pathways and the phosphorylation cascade which are involved in the protection of B cell from apoptosis after treatment with IFN-alpha/beta. Copyright 2010 Elsevier Inc. All rights reserved.

Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

PubMed Central

Phatak, Nitasha R.; Stankowska, Dorota L.

2016-01-01

Purpose Brn3b is a class IV POU domain transcription factor that plays an important role in the development of retinal ganglion cells (RGCs), RGC survival, and particularly axon growth and pathfinding. Our previous study demonstrated that recombinant adenoassociated virus serotype 2 (rAAV-2)–mediated overexpression of Brn3b in RGCs promoted neuroprotection in a rodent model of glaucoma. However, the mechanisms underlying neuroprotection of RGCs in rats overexpressing Brn3b in animal models of glaucoma remain largely unknown. The goal of this study was to understand some of the mechanisms underlying the neuroprotection of RGCs overexpressing Brn3b during intraocular pressure (IOP) elevation in Brown Norway rats. Methods One eye of Brown Norway rats (Rattus norvegicus) was injected with an AAV construct encoding either green fluorescent protein (GFP; recombinant adenoassociated virus–green fluorescent protein, rAAV-hSyn-GFP) or Brn3b (rAAV-hSyn-Brn3b). Expression of antiapoptotic proteins, including B cell lymphoma/leukemia-2 (Bcl-2) family proteins (Bcl-2 and Bcl-xL), and p-AKT, was observed following immunostaining of rat retinas that overexpress Brn3b. In a different set of experiments, intraocular pressure was elevated in one eye of Brown Norway rats, which was followed by intravitreal injection with AAV constructs encoding either GFP (rAAV-CMV-GFP) or Brn3b (rAAV-CMV-Brn3b). Retinal sections were stained for prosurvival factors, including Bcl-2, Bcl-XL, and p-AKT. Results AAV-mediated expression of transcription factor Brn3b promoted statistically significant upregulation of the Bcl-2 protein and increased expression of p-AKT in RGCs of Brown Norway rats. In addition, following IOP elevation, AAV-mediated Brn3b expression also statistically significantly increased levels of Bcl-2 in the RGC layer in Brown Norway rats. Conclusions Adenoassociated virus–mediated Brn3b protein overexpression may promote neuroprotection by upregulating key antiapoptotic

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

PubMed Central

2012-01-01

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. Methods The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. Results In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. Conclusion Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors. PMID:23171055

Attacking Cancer’s Achilles Heel: Antagonism of Anti-Apoptotic BCL-2 Family Members

PubMed Central

Opferman, Joseph T.

2015-01-01

Malignant cells routinely violate cellular checkpoints that should initiate cell death in normal cells by triggering pro-apoptotic members of the BCL-2 family of proteins. To escape such death inducing signals, cancer cells often select for up regulation of anti-apoptotic BCL-2 family members including BCL-2, BCL-XL, BFL-1, BCL-W, and MCL-1. These family members prevent death by sequestering pro-apoptotic molecules. To counter this resistance mechanism, small molecule inhibitors of anti-apoptotic BCL-2 family members have been under development. These molecules have shown promise in pre-clinical and clinical testing to overcome apoptotic resistance, prompting cancer cells to undergo apoptosis. Alternatively, other strategies have taken advantage of the normal regulatory machinery controlling anti-apoptotic molecules and have used inhibitors of signaling pathways to down-modulate the expression of anti-apoptotic molecules thus tilting the balance in cancer cells to cell death. This review explores recent developments and strategies aimed at antagonizing anti-apoptotic BCL-2 family member action to promote the induction of cell death in cancer therapy. PMID:26293580

Systems modeling accurately predicts responses to genotoxic agents and their synergism with BCL-2 inhibitors in triple negative breast cancer cells.

PubMed

Lucantoni, Federico; Lindner, Andreas U; O'Donovan, Norma; Düssmann, Heiko; Prehn, Jochen H M

2018-01-19

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer which accounts for 15-20% of this disease and is currently treated with genotoxic chemotherapy. The BCL2 (B-cell lymphoma 2) family of proteins controls the process of mitochondrial outer membrane permeabilization (MOMP), which is required for the activation of the mitochondrial apoptosis pathway in response to genotoxic agents. We previously developed a deterministic systems model of BCL2 protein interactions, DR_MOMP that calculates the sensitivity of cells to undergo mitochondrial apoptosis. Here we determined whether DR_MOMP predicts responses of TNBC cells to genotoxic agents and the re-sensitization of resistant cells by BCL2 inhibitors. Using absolute protein levels of BAX, BAK, BCL2, BCL(X)L and MCL1 as input for DR_MOMP, we found a strong correlation between model predictions and responses of a panel of TNBC cells to 24 and 48 h cisplatin (R 2  = 0.96 and 0.95, respectively) and paclitaxel treatments (R 2  = 0.94 and 0.95, respectively). This outperformed single protein correlations (best performer BCL(X)L with R 2 of 0.69 and 0.50 for cisplatin and paclitaxel treatments, respectively) and BCL2 proteins ratio (R 2 of 0.50 for cisplatin and 0.49 for paclitaxel). Next we performed synergy studies using the BCL2 selective antagonist Venetoclax /ABT199, the BCL(X)L selective antagonist WEHI-539, or the MCL1 selective antagonist A-1210477 in combination with cisplatin. In silico predictions by DR_MOMP revealed substantial differences in treatment responses of BCL(X)L, BCL2 or MCL1 inhibitors combinations with cisplatin that were successfully validated in cell lines. Our findings provide evidence that DR_MOMP predicts responses of TNBC cells to genotoxic therapy, and can aid in the choice of the optimal BCL2 protein antagonist for combination treatments of resistant cells.

Multiple functions of BCL-2 family proteins.

PubMed

Hardwick, J Marie; Soane, Lucian

2013-02-01

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.

Homology modeling and docking studies of human Bcl-2L10 protein.

PubMed

Bhargavi, K; Kalyan Chaitanya, P; Ramasree, D; Vasavi, M; Murthy, D K; Uma, V

2010-12-01

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B - Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti-apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein - protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro-apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.

The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination

PubMed Central

Lee, Baeck-seung; Dekker, Joseph D.; Lee, Bum-kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.

2013-01-01

Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11alox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:23438597

BCL-W has a fundamental role in B cell survival and lymphomagenesis.

PubMed

Adams, Clare M; Kim, Annette S; Mitra, Ramkrishna; Choi, John K; Gong, Jerald Z; Eischen, Christine M

2017-02-01

Compromised apoptotic signaling is a prerequisite for tumorigenesis. The design of effective therapies for cancer treatment depends on a comprehensive understanding of the mechanisms that govern cell survival. The antiapoptotic proteins of the BCL-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Unlike BCL-2 and BCL-XL, the closest antiapoptotic relative BCL-W is required for spermatogenesis, but was considered dispensable for all other cell types. Here, however, we have exposed a critical role for BCL-W in B cell survival and lymphomagenesis. Loss of Bcl-w conferred sensitivity to growth factor deprivation-induced B cell apoptosis. Moreover, Bcl-w loss profoundly delayed MYC-mediated B cell lymphoma development due to increased MYC-induced B cell apoptosis. We also determined that MYC regulates BCL-W expression through its transcriptional regulation of specific miR. BCL-W expression was highly selected for in patient samples of Burkitt lymphoma (BL), with 88.5% expressing BCL-W. BCL-W knockdown in BL cell lines induced apoptosis, and its overexpression conferred resistance to BCL-2 family-targeting BH3 mimetics. Additionally, BCL-W was overexpressed in diffuse large B cell lymphoma and correlated with decreased patient survival. Collectively, our results reveal that BCL-W profoundly contributes to B cell lymphoma, and its expression could serve as a biomarker for diagnosis and aid in the development of better targeted therapies.

BCL-W has a fundamental role in B cell survival and lymphomagenesis

PubMed Central

Adams, Clare M.; Kim, Annette S.; Mitra, Ramkrishna; Choi, John K.; Gong, Jerald Z.; Eischen, Christine M.

2017-01-01

Compromised apoptotic signaling is a prerequisite for tumorigenesis. The design of effective therapies for cancer treatment depends on a comprehensive understanding of the mechanisms that govern cell survival. The antiapoptotic proteins of the BCL-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Unlike BCL-2 and BCL-XL, the closest antiapoptotic relative BCL-W is required for spermatogenesis, but was considered dispensable for all other cell types. Here, however, we have exposed a critical role for BCL-W in B cell survival and lymphomagenesis. Loss of Bcl-w conferred sensitivity to growth factor deprivation–induced B cell apoptosis. Moreover, Bcl-w loss profoundly delayed MYC-mediated B cell lymphoma development due to increased MYC-induced B cell apoptosis. We also determined that MYC regulates BCL-W expression through its transcriptional regulation of specific miR. BCL-W expression was highly selected for in patient samples of Burkitt lymphoma (BL), with 88.5% expressing BCL-W. BCL-W knockdown in BL cell lines induced apoptosis, and its overexpression conferred resistance to BCL-2 family–targeting BH3 mimetics. Additionally, BCL-W was overexpressed in diffuse large B cell lymphoma and correlated with decreased patient survival. Collectively, our results reveal that BCL-W profoundly contributes to B cell lymphoma, and its expression could serve as a biomarker for diagnosis and aid in the development of better targeted therapies. PMID:28094768

BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).

PubMed

Maiuri, Maria Chiara; Criollo, Alfredo; Tasdemir, Ezgi; Vicencio, José Miguel; Tajeddine, Nicolas; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-01-01

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.

microRNAs affect BCL-2 family proteins in the setting of cerebral ischemia

PubMed Central

Ouyang, Yi-Bing; Giffard, Rona G.

2014-01-01

The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca2+ from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNA), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins. PMID:24373752

MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia.

PubMed

Ouyang, Yi-Bing; Giffard, Rona G

2014-11-01

The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation

PubMed Central

Anderson, Grace R.; Wardell, Suzanne E.; Cakir, Merve; Crawford, Lorin; Leeds, Jim C.; Nussbaum, Daniel P.; Shankar, Pallavi S.; Soderquist, Ryan S.; Stein, Elizabeth M.; Tingley, Jennifer P.; Winter, Peter S.; Zieser-Misenheimer, Elizabeth K.; Alley, Holly M.; Yllanes, Alexander; Haney, Victoria; Blackwell, Kimberly L.; McCall, Shannon J.; McDonnell, Donald P.; Wood, Kris C.

2017-01-01

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that the combined inhibition of BCL-XL and the mTOR/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses MCL-1 protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-XL. This dual dependence on BCL-XL and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of molecular subtype or PIK3CA mutational status. Further, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-XL/MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to anti-apoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-XL also sensitizes breast cancer cells to standard of care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-XL and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-XL may be effective in tumors lacking PIK3CA mutations. PMID:27974663

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL.

PubMed

Liu, Xiaorong; Beugelsdijk, Alex; Chen, Jianhan

2015-09-01

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells*

PubMed Central

Smith, Alyson J.; Dai, Haiming; Correia, Cristina; Takahashi, Rie; Lee, Sun-Hee; Schmitz, Ingo; Kaufmann, Scott H.

2011-01-01

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (KD) of 3.4 nm for Mcl-1, 70 nm for Bcl-xL, and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-xL antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance. PMID:21454712

CASP10-BCL::Fold efficiently samples topologies of large proteins.

PubMed

Heinze, Sten; Putnam, Daniel K; Fischer, Axel W; Kohlmann, Tim; Weiner, Brian E; Meiler, Jens

2015-03-01

During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein. © 2015 Wiley Periodicals, Inc.

ProXL (Protein Cross-Linking Database): A Platform for Analysis, Visualization, and Sharing of Protein Cross-Linking Mass Spectrometry Data

PubMed Central

2016-01-01

ProXL is a Web application and accompanying database designed for sharing, visualizing, and analyzing bottom-up protein cross-linking mass spectrometry data with an emphasis on structural analysis and quality control. ProXL is designed to be independent of any particular software pipeline. The import process is simplified by the use of the ProXL XML data format, which shields developers of data importers from the relative complexity of the relational database schema. The database and Web interfaces function equally well for any software pipeline and allow data from disparate pipelines to be merged and contrasted. ProXL includes robust public and private data sharing capabilities, including a project-based interface designed to ensure security and facilitate collaboration among multiple researchers. ProXL provides multiple interactive and highly dynamic data visualizations that facilitate structural-based analysis of the observed cross-links as well as quality control. ProXL is open-source, well-documented, and freely available at https://github.com/yeastrc/proxl-web-app. PMID:27302480

ProXL (Protein Cross-Linking Database): A Platform for Analysis, Visualization, and Sharing of Protein Cross-Linking Mass Spectrometry Data.

PubMed

Riffle, Michael; Jaschob, Daniel; Zelter, Alex; Davis, Trisha N

2016-08-05

ProXL is a Web application and accompanying database designed for sharing, visualizing, and analyzing bottom-up protein cross-linking mass spectrometry data with an emphasis on structural analysis and quality control. ProXL is designed to be independent of any particular software pipeline. The import process is simplified by the use of the ProXL XML data format, which shields developers of data importers from the relative complexity of the relational database schema. The database and Web interfaces function equally well for any software pipeline and allow data from disparate pipelines to be merged and contrasted. ProXL includes robust public and private data sharing capabilities, including a project-based interface designed to ensure security and facilitate collaboration among multiple researchers. ProXL provides multiple interactive and highly dynamic data visualizations that facilitate structural-based analysis of the observed cross-links as well as quality control. ProXL is open-source, well-documented, and freely available at https://github.com/yeastrc/proxl-web-app .

Targeting BCL-2-like Proteins to Kill Cancer Cells.

PubMed

Cory, Suzanne; Roberts, Andrew W; Colman, Peter M; Adams, Jerry M

2016-08-01

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

PubMed

Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

2016-11-02

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

FBXO10 deficiency and BTK activation upregulate BCL2 expression in mantle cell lymphoma.

PubMed

Li, Y; Bouchlaka, M N; Wolff, J; Grindle, K M; Lu, L; Qian, S; Zhong, X; Pflum, N; Jobin, P; Kahl, B S; Eickhoff, J C; Wuerzberger-Davis, S M; Miyamoto, S; Thomas, C J; Yang, D T; Capitini, C M; Rui, L

2016-12-01

Targeting Bruton tyrosine kinase (BTK) by ibrutinib is an effective treatment for patients with relapsed/refractory mantle cell lymphoma (MCL). However, both primary and acquired resistance to ibrutinib have developed in a significant number of these patients. A combinatory strategy targeting multiple oncogenic pathways is critical to enhance the efficacy of ibrutinib. Here, we focus on the BCL2 anti-apoptotic pathway. In a tissue microarray of 62 MCL samples, BCL2 expression positively correlated with BTK expression. Increased levels of BCL2 were shown to be due to a defect in protein degradation because of no or little expression of the E3 ubiquitin ligase FBXO10, as well as transcriptional upregulation through BTK-mediated canonical nuclear factor-κB activation. RNA-seq analysis confirmed that a set of anti-apoptotic genes (for example, BCL2, BCL-XL and DAD1) was downregulated by BTK short hairpin RNA. The downregulated genes also included those that are critical for B-cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Targeting BCL2 by the specific inhibitor ABT-199 synergized with ibrutinib in inhibiting growth of both ibrutinib-sensitive and -resistant cancer cells in vitro and in vivo. These results suggest co-targeting of BTK and BCL2 as a new therapeutic strategy in MCL, especially for patients with primary resistance to ibrutinib.

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

PubMed Central

McClintock, David S.; Santore, Matthew T.; Lee, Vivian Y.; Brunelle, Joslyn; Budinger, G. R. Scott; Zong, Wei-Xing; Thompson, Craig B.; Hay, Nissim; Chandel, Navdeep S.

2002-01-01

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-XL prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-XL to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F1F0-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-XL, cells devoid of mitochondrial DNA (ρ° cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax−/− bak−/− mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation. PMID:11739725

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

PubMed Central

Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

2016-01-01

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. DOI: http://dx.doi.org/10.7554/eLife.20352.001 PMID:27805565

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

PubMed

Aouacheria, Abdel; Baghdiguian, Stephen; Lamb, Heather M; Huska, Jason D; Pineda, Fernando J; Hardwick, J Marie

2017-10-01

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expanding the Cancer Arsenal with Targeted Therapies: Disarmament of the Antiapoptotic Bcl-2 Proteins by Small Molecules.

PubMed

Yap, Jeremy L; Chen, Lijia; Lanning, Maryanna E; Fletcher, Steven

2017-02-09

A hallmark of cancer is the evasion of apoptosis, which is often associated with the upregulation of the antiapoptotic members of the Bcl-2 family of proteins. The prosurvival function of the antiapoptotic Bcl-2 proteins is manifested by capturing and neutralizing the proapoptotic Bcl-2 proteins via their BH3 death domains. Accordingly, strategies to antagonize the antiapoptotic Bcl-2 proteins have largely focused on the development of low-molecular-weight, synthetic BH3 mimetics ("magic bullets") to disrupt the protein-protein interactions between anti- and proapoptotic Bcl-2 proteins. In this way, apoptosis has been reactivated in malignant cells. Moreover, several such Bcl-2 family inhibitors are presently being evaluated for a range of cancers in clinical trials and show great promise as new additions to the cancer armamentarium. Indeed, the selective Bcl-2 inhibitor venetoclax (Venclexta) recently received FDA approval for the treatment of a specific subset of patients with chronic lymphocytic leukemia. This review focuses on the major developments in the field of Bcl-2 inhibitors over the past decade, with particular emphasis on binding modes and, thus, the origins of selectivity for specific Bcl-2 family members.

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

PubMed

Oldereid, N B; Angelis, P D; Wiger, R; Clausen, O P

2001-05-01

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

The AA genotype of the regulatory BCL2 promoter polymorphism ( 938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients.

PubMed

Bachmann, Hagen S; Otterbach, Friedrich; Callies, Rainer; Nückel, Holger; Bau, Maja; Schmid, Kurt W; Siffert, Winfried; Kimmig, Rainer

2007-10-01

Expression of the antiapoptotic and antiproliferative protein Bcl-2 has been repeatedly shown to be associated with better clinical outcome in breast cancer. We recently showed a novel regulatory (-938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter generating significantly different BCL2 promoter activities. Paraffin-embedded neoplastic and nonneoplastic tissues from 274 patients (161 still alive after a follow-up period of at least 80 months) with primary unilateral invasive breast carcinoma were investigated. Bcl-2 expression of tumor cells was shown by immunohistochemistry; nonneoplastic tissues were used for genotyping. Both the Bcl-2 expression and the (-938C>A) genotypes were correlated with the patients' survival. Kaplan-Meier curves revealed a significant association of the AA genotype with increased survival (P = 0.030) in lymph node-negative breast cancer patients, whereas no genotype effect could be observed in lymph node-positive cases. Ten-year survival rates were 88.6% for the AA genotype, 78.4% for the AC genotype, and 65.8% for the CC genotype. Multivariable Cox regression identified the BCL2 (-938CC) genotype as an independent prognostic factor for cancer-related death in lymph node-negative breast carcinoma patients (hazard ratio, 3.59; P = 0.032). Immunohistochemical Bcl-2 expression was significantly associated with the clinical outcome of lymph node-positive but not of lymph node-negative breast cancer patients. In lymph node-negative cases, the (-938C>A) SNP was both significantly related with the immunohistochemically determined level of Bcl-2 expression (P = 0.044) and the survival of patients with Bcl-2-expressing carcinomas (P = 0.006). These results suggest the (-938C>A) polymorphism as a survival prognosticator as well as indicator of a high-risk group within patients with lymph node-negative breast cancer.

Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

PubMed Central

DeBartolo, Joe; Taipale, Mikko; Keating, Amy E.

2014-01-01

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-xL and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models. PMID:24967846

p38 mitogen-activated protein kinase-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury.

PubMed

Jiang, Shao-Yun; Zou, Yuan-Yuan; Wang, Jian-Tao

2012-01-01

In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. These findings provide evidence of crosstalk between p38 MAPK and NF-κB p65 and

p38 mitogen-activated protein kinase–induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury

PubMed Central

Jiang, Shao-Yun; Zou, Yuan-Yuan

2012-01-01

Purpose In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Methods Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. Results The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. Conclusions These findings provide evidence of crosstalk

First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.

PubMed

Boidol, Bernd; Kornauth, Christoph; van der Kouwe, Emiel; Prutsch, Nicole; Kazianka, Lukas; Gültekin, Sinan; Hoermann, Gregor; Mayerhoefer, Marius E; Hopfinger, Georg; Hauswirth, Alexander; Panny, Michael; Aretin, Marie-Bernadette; Hilgarth, Bernadette; Sperr, Wolfgang R; Valent, Peter; Simonitsch-Klupp, Ingrid; Moriggl, Richard; Merkel, Olaf; Kenner, Lukas; Jäger, Ulrich; Kubicek, Stefan; Staber, Philipp B

2017-12-07

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy usually refractory to current treatment strategies and associated with short overall survival. By applying next-generation functional testing of primary patient-derived lymphoma cells using a library of 106 US Food and Drug Administration (FDA)-approved anticancer drugs or compounds currently in clinical development, we set out to identify novel effective treatments for T-PLL patients. We found that the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (ABT-199) demonstrated the strongest T-PLL-specific response when comparing individual ex vivo drug response in 86 patients with refractory hematologic malignancies. Mechanistically, responses to venetoclax correlated with protein expression of BCL-2 but not with expression of the BCL-2 family members myeloid cell leukemia 1 (MCL-1) and BCL-XL in lymphoma cells. BCL-2 expression was inversely correlated with the expression of MCL-1. Based on the ex vivo responses, venetoclax treatment was commenced in 2 late-stage refractory T-PLL patients resulting in clinical responses. Our findings demonstrate first evidence of single-agent activity of venetoclax both ex vivo and in humans, offering a novel agent in T-PLL. © 2017 by The American Society of Hematology.

A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

PubMed

Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu

2016-01-01

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins.

PubMed

Weber, Arnim; Paschen, Stefan A; Heger, Klaus; Wilfling, Florian; Frankenberg, Tobias; Bauerschmitt, Heike; Seiffert, Barbara M; Kirschnek, Susanne; Wagner, Hermann; Häcker, Georg

2007-05-21

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.

Combined Targeting of BCL-2 and BCR-ABL Tyrosine Kinase Eradicates Chronic Myeloid Leukemia Stem Cells

PubMed Central

Mak, Po Yee; Mu, Hong; Zhou, Hongsheng; Mak, Duncan H.; Schober, Wendy; Leverson, Joel D.; Zhang, Bin; Bhatia, Ravi; Huang, Xuelin; Cortes, Jorge; Kantarjian, Hagop; Konopleva, Marina

2016-01-01

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML. PMID:27605552

Simvastatin induces derepression of PTEN expression via NFkappaB to inhibit breast cancer cell growth.

PubMed

Ghosh-Choudhury, Nayana; Mandal, Chandi Charan; Ghosh-Choudhury, Nandini; Ghosh Choudhury, Goutam

2010-05-01

Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which causes tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of the expression of the anti-apoptotic protein Bcl(XL). In many cancer cells, Bcl(XL) is a target of NFkappaB. Simvastatin inhibited the DNA binding and transcriptional activities of NFkappaB resulting in marked reduction in transcription of Bcl(XL). Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFkappaB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of Bcl(XL), simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFkappaB p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFkappaB, which attenuates the expression of anti-apoptotic Bcl(XL) and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth. Published by Elsevier Inc.

Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

PubMed

Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

2015-09-01

Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

PubMed

Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

2018-04-25

Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

PubMed

Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

2016-04-07

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential.

[Distribution of Pathogenic Bacteria and Its Influence on Expression of BCL-2 and BAX Protein after HSCT in the Patients with Hematological Malignancies].

PubMed

Su, Gui-Ping; Dai, Yan; Huang, Lai-Quan; Jiang, Yi-Zhi; Geng, Liang-Quan; Ding, Kai-Yang; Huang, Dong-Ping

2016-06-01

To investigate the distribution of pathogenic bacteria in the patients with hematologic malignancies received hematopoietic stem cell transplantation (HSCT) and its influence on the expression of BCL-2 and BAX proteins. The clinical data of 64 patients with malignant lymphoma (ML) received auto-HSCT from January 2011 to December 2015 in our hospital were analyzed. On basis of post-treansplant infection, the patients were divided into infection group (36 cases) and non-infection group (28 cases). The distribution of pathogenic bacteria in 2 groups was identified, the T lymphocyte subsets of peripheral blood, expression level of apoptotic proteins and C-reaction protein (CRP) in 2 group were detected. Thirty-six strains of pathogenic bacteria were isolated from 36 case of hematological malignancy after HSCT, including 24 strains of Gram-negative bacteria (66.67%) with predominamce of klebsiella pneumoniae (19.44%). The periperal blood CD4+ (t=2.637, P<0.01), CD4+/CD8+ ratio (t=8.223, P<0.01), BCL-2 protein (t=5.852, P<0.05), BCL-2/BAX ratio (t=14.56, P<0.01) in infection group were significantly lower than those in non-infection group, while CD8+ (t=2.285, P=<0.01), CRP (t=39.71, P<0.01), BAX level in infection group were higher than those in non-infection group. The pearson correcation analysis showed that the CD4+/CD8+ ratio in infection group positively correlated with BCL-2/BAX ratio (t=0.341, P<0.05), while serum CRP level in infection group negatively correlated with BCL-2/BAX ratio (t=-0.362, P<0.05). The pathogenic bacteria infecting ML patients after HSCT were mainly Gram-negative bacteria. The post-transplant infection can promote the expression up-regulation of related inflammatory factors and apoptotic proteins. The pathogens may be involved in cell apoptisis that provides a new strategy to treat the hematologic malignancies.

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner.

PubMed

Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D; Wang, Hong-Gang; Sebti, Saïd M

2011-03-18

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Connexin 26 correlates with Bcl-xL and Bax proteins expression in colorectal cancer

PubMed Central

Kanczuga-Koda, Luiza; Sulkowski, Stanislaw; Koda, Mariusz; Skrzydlewska, Elzbieta; Sulkowska, Mariola

2005-01-01

AIM: To evaluate of Cx26 in correlation with Bcl-xL and Bax proteins in colorectal cancer. METHODS: Immunohistochemical staining using specific antibodies was performed to evaluate the protein expression of Cx26, Bax and Bcl-xL in 152 colorectal cancer samples and the correlations among studied proteins as well as the relationships between the expression of Cx26, Bax, Bcl-xL and clinicopathological features were analyzed. RESULTS: Both normal epithelial cells and carcinoma cells expressed Cx26, Bax and Bcl-xL, but Cx26 in cancer cells showed aberrant, mainly cytoplasmic staining. Expression of Cx26, Bax and Bcl-xL was observed in 55.9%, 55.5% and 72.4% of evaluated colorectal cancers respectively. We found the positive correlation between Cx26 and Bax expression (r = 0.561, P<0.0001), Cx26 and Bcl-xL (r = 0.409, P<0.0001) as well as between Bax and Bcl-xL (r = 0.486, P<0.0001). Association of Cx26, Bax and Bcl-xL expression with histological G2 grade of tumors was noted (P<0.005, P<0.001 and P<0.002 respectively). CONCLUSION: Cytoplasmic presence of Cx26 and its association with apoptotic markers could indicate a distinct role from physiological functions of Cx26 in cancer cells and it could suggest that connexins might be a target point for modulations of apoptosis with therapeutic implications. PMID:15770735

Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

PubMed

Wallgren, Marcus; Lidman, Martin; Pedersen, Anders; Brännström, Kristoffer; Karlsson, B Göran; Gröbner, Gerhard

2013-01-01

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

Effect of hypoxia on the expression of pro- and anti-apoptotic proteins in neuronal nuclei of the guinea pig fetus during gestation.

PubMed

Abedin, Naheed; Ashraf, Qazi; Mishra, Om Prakash; Delivoria-Papadopoulos, Maria

2005-04-21

The present study investigates the expression of apoptotic proteins Bax, Bad, Bcl-2, and Bcl-xl following hypoxia in the cerebral cortex of the guinea pig fetus as a function of gestational age. Normoxic (Nx, n = 6) and hypoxic (Hx, n = 6) guinea pig fetuses at 35 and 60 days gestation were studied. Bax expression (OD X mm(2)) was 96.9 +/- 9.5 (Nx 35 days), 116.5 +/- 8.3 (Hx 35 days), P < 0.05 and 116.2 +/- 3.4 (Nx 60 days, 144.6 +/- 11.7 (Hx 60 days), P < 0.05. Bad expression (OD X mm(2)) was 78.6 +/- 2.6 (Nx 35 days), 102.9 +/- 5.8 (Hx 35 days), P < 0.05 and 101.5 +/- 4.3 (Nx 60 days), 139.8 +/- 7.9 (Hx 60 days), P < 0.05 vs. Nx 60 days, also significantly higher from preterm hypoxia P < 0.007. Expression of Bcl-2 (OD X mm(2)) was 27.4 +/- 2.0 (Nx 35 days), 28.0 +/- 2.4 (Hx 35 days), and 27.4 +/- 2.7 (Nx 60 days), 29.7 +/- 2.3 (Hx 60 days). Expression of Bcl-xl (OD X mm(2)) was 51.0 +/- 4.4 (Nx 35 days), 46.1 +/- 8.0 (Hx 35 days) and 50.0 +/- 1.4 (Nx 60 days), 54.9 +/- 7.4 (Hx 60 days). Hypoxia resulted in increased expression of the proapoptotic proteins Bax and Bad by 20% and 30% in the preterm as compared to 24% and 38% at term, without altering the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. We conclude that the hypoxia-induced increased expression of Bax and Bad is greater at term compared to preterm. Furthermore, the hypoxia-induced increase in proapoptotic as compared to antiapoptotic proteins at term will accelerate the ongoing active process of programmed cell death at term compared to preterm gestation.

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages.

PubMed

Sukumaran, Sunil K; Selvaraj, Suresh K; Prasadarao, Nemani V

2004-10-01

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.

Propofol-induced rno-miR-665 targets BCL2L1 and influences apoptosis in rodent developing hippocampal astrocytes.

PubMed

Sun, Wen-Chong; Liang, Zuo-Di; Pei, Ling

2015-12-01

Propofol exerts neurotoxic effects on the developing mammalian brains, but the underlying molecular mechanism remains unclear. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, in specific types of neurocytes, the detailed functions of miRNAs were not entirely understood. We investigated the potential role of miRNAs in astrocyte pathogenesis caused by propofol. We performed genome-wide microRNA expression profiling in immature cultured hippocampal astrocytes by microarray analysis and predicted their targets and functions using bioinformatics tools. The functional effects of one differentially expressed miRNA were examined experimentally in relation to astrocyte viability. The results showed that 13 miRNAs were significantly differentially expressed after both short-term exposure to high-concentration propofol (10 μg/ml for 1h) and long-term exposure to low-concentration propofol (0.9 μg/ml for 48 h), including rno-miR-665, differing significantly between the 2. Bioinformatics predicted putative binding sites for rno-miR-665 existing in the 3'-untranslated region of Bcl-2-like protein 1 BCL2L1 (Bcl-xl) mRNA. Moreover, such relationship was assessed by luciferase reporter assay, qRT-PCR and western blot. Rno-miR-665 which was significantly up-regulated by propofol can suppress BCL2L1 and elevate cleaved caspase-3 expression in immature astrocytes in vitro. Apoptosis of developing hippocampal astrocytes was thus significantly influenced by propofol or rno-miR-665, or both. Taken together, rno-miR-665 is involved in the neurotoxicity induced by propofol via a caspase-3 mediated mechanism by negatively regulating BCL2L1. It might act as an alternative therapeutic target for treatment of neurological disorders in peadiatric prolonged anesthesia or sedation with propofol clinically. Copyright © 2015. Published by Elsevier B.V.

High levels of bcl-2 protein expression do not correlate with genetic abnormalities but predict worse prognosis in patients with lymphoblastic lymphoma.

PubMed

Gu, Yajun; Pan, Yi; Meng, Bin; Guan, Bingxin; Fu, Kai; Sun, Baocun; Zheng, Fang

2013-06-01

We aimed to investigate bcl-2, bcl-6, and c-myc rearrangements in patients with lymphoblastic lymphoma (LBL), especially focus on the correlation of protein expression with genetic abnormalities. Moreover, their prognostic significance was further analyzed in LBL. Protein expression and genetic abnormalities of bcl-2, bcl-6, and c-myc were investigated in microarrayed tumors from 33 cases of T cell LBL and eight cases of B cell lineage. Immunohistochemical (IHC) staining was performed to evaluate protein expression, including bcl-2, bcl-6, c-myc, TdT, CD1α, CD34, Ki-67, PAX-5, CD2, CD3, CD4, CD8, and CD20. Genetic abnormalities of bcl-2, bcl-6, and c-myc were detected by dual color fluorescence in situ hybridization (FISH). Bcl-2 protein was positive in 51.2 % (21/41) of the patients, bcl-6 protein in 7.3 % (three out of 41), and c-myc protein in 78.0 % (32/41). Bcl-2 breakpoint was found in two cases by FISH analysis. There was no evidence of bcl-6 or c-myc rearrangement in patients with LBL. However, both gene gain and loss events occurred in bcl-2, bcl-6, and c-myc. A univariate analysis showed that stage III or IV, elevated lactate dehydrogenase (LDH), and positivity for bcl-2 protein were associated with shorter survival (p<0.05). Enhanced protein expression and detectable genetic abnormalities of bcl-2, bcl-6, and c-myc were observed in patients with LBL. No statistical correlation was found between IHC results and cytogenetic findings. Stage III or IV, elevated LDH, and positivity for bcl-2 protein were identified as adverse prognostic factors. The patients with more adverse factors would have increasingly worse prognosis.

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

PubMed

Renault, Thibaud T; Elkholi, Rana; Bharti, Archana; Chipuk, Jerry E

2014-09-19

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Grouper iridovirus GIV66 is a Bcl-2 protein that inhibits apoptosis by exclusively sequestering Bim.

PubMed

Banjara, Suresh; Mao, Jiahao; Ryan, Timothy M; Caria, Sofia; Kvansakul, Marc

2018-04-13

Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode functional and structural homologs of Bcl-2 to counter premature host-cell apoptosis and ensure viral proliferation or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the Iridoviridae family and harbors GIV66, a putative Bcl-2-like protein and mitochondrially localized apoptosis inhibitor. However, the molecular and structural basis of GIV66-mediated apoptosis inhibition is currently not understood. To gain insight into GIV66's mechanism of action, we systematically evaluated its ability to bind peptides spanning the BH3 domain of pro-apoptotic Bcl-2 family members. Our results revealed that GIV66 harbors an unusually high level of specificity for pro-apoptotic Bcl-2 and displays affinity only for Bcl-2-like 11 (Bcl2L11 or Bim). Using crystal structures of both apo-GIV66 and GIV66 bound to the BH3 domain from Bim, we unexpectedly found that GIV66 forms dimers via an interface that results in occluded access to the canonical Bcl-2 ligand-binding groove, which breaks apart upon Bim binding. This observation suggests that GIV66 dimerization may affect GIV66's ability to bind host pro-death Bcl-2 proteins and enables highly targeted virus-directed suppression of host apoptosis signaling. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single-specificity, pro-survival Bcl-2 protein and identifying a pivotal role of Bim in GIV-mediated inhibition of apoptosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Low rate of apoptosis and overexpression of bcl-2 in Epstein-Barr virus-associated gastric carcinoma.

PubMed

Kume, T; Oshima, K; Shinohara, T; Takeo, H; Yamashita, Y; Shirakusa, T; Kikuchi, M

1999-06-01

Epstein-Barr virus (EBV) has been demonstrated in about 10% of gastric carcinomas. However, the pathogenetic role of EBV in gastric carcinoma is uncertain. We compared the rate of apoptotic cell death, cell proliferation and the expression of apoptosis-related proteins in gastric carcinomas with or without EBV. Epstein-Barr virus was detected in 40 gastric carcinomas by EBV-encoded small RNA-1 in-situ hybridization. Apoptotic cell death, MIB-1, p53, bcl-2 and bcl-x were examined by the terminal deoxynucleotidyl-mediated dUTP-nick end labelling method and immunohistochemistry. We also included 40 age-, sex- and disease stage-matched EBV-negative cases as a control. The number of apoptotic cells was significantly lower in EBV-positive (20 +/- 15. 1/1000 cells) and bcl-2-positive (17 +/- 12.9/1000 cells) tumours than in EBV-negative (43 +/- 37.1) and bcl-2-negative tumours (38 +/- 32.1, P < 0.001, P < 0.001, respectively). bcl-2 immunostaining was significantly higher in EBV-positive tumours (24 cases) than in EBV-negative tumours (12 cases, P < 0.05). There was no significant difference in bcl-x and p53 expression between EBV-positive and -negative tumours. The number of MIB-1-positive cells in EBV-positive tumours (237 +/- 161/1000) was significantly lower than in EBV-negative tumours (480 +/- 208/1000 cells, P < 0.001). A low rate of apoptosis and high bcl-2 expression were recognized in EBV-positive gastric carcinomas, suggesting that bcl-2 protein is the main inhibitor of apoptosis in EBV-positive carcinomas. In addition, the low apoptotic and proliferative activities may reflect a low biological activity in EBV-positive gastric carcinomas.

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain.

PubMed

Raghav, Pawan Kumar; Verma, Yogesh Kumar; Gangenahalli, Gurudutta U

2012-05-01

B-cell lymphoma (Bcl-2) protein is an anti-apoptotic member of the Bcl-2 family. It is functionally demarcated into four Bcl-2 homology (BH) domains: BH1, BH2, BH3, BH4, one flexible loop domain (FLD), a transmembrane domain (TM), and an X domain. Bcl-2's BH domains have clearly been elucidated from a structural perspective, whereas the conformation of FLD has not yet been predicted, despite its important role in regulating apoptosis through its interactions with JNK-1, PKC, PP2A phosphatase, caspase 3, MAP kinase, ubiquitin, PS1, and FKBP38. Many important residues that regulate Bcl-2 anti-apoptotic activity are present in this domain, for example Asp34, Thr56, Thr69, Ser70, Thr74, and Ser87. The structural elucidation of the FLD would likely help in attempts to accurately predict the effect of mutating these residues on the overall structure of the protein and the interactions of other proteins in this domain. Therefore, we have generated an increased quality model of the Bcl-2 protein including the FLD through modeling. Further, molecular dynamics (MD) simulations were used for FLD optimization, to predict the flexibility, and to determine the stability of the folded FLD. In addition, essential dynamics (ED) was used to predict the collective motions and the essential subspace relevant to Bcl-2 protein function. The predicted average structure and ensemble of MD-simulated structures were submitted to the Protein Model Database (PMDB), and the Bcl-2 structures obtained exhibited enhanced quality. This study should help to elucidate the structural basis for Bcl-2 anti-apoptotic activity regulation through its binding to other proteins via the FLD.

Structural changes in the BH3 domain of SOUL protein upon interaction with the anti-apoptotic protein Bcl-xL

PubMed Central

Ambrosi, Emmanuele; Capaldi, Stefano; Bovi, Michele; Saccomani, Gianmaria; Perduca, Massimiliano; Monaco, Hugo L.

2011-01-01

The SOUL protein is known to induce apoptosis by provoking the mitochondrial permeability transition, and a sequence homologous with the BH3 (Bcl-2 homology 3) domains has recently been identified in the protein, thus making it a potential new member of the BH3-only protein family. In the present study, we provide NMR, SPR (surface plasmon resonance) and crystallographic evidence that a peptide spanning residues 147–172 in SOUL interacts with the anti-apoptotic protein Bcl-xL. We have crystallized SOUL alone and the complex of its BH3 domain peptide with Bcl-xL, and solved their three-dimensional structures. The SOUL monomer is a single domain organized as a distorted β-barrel with eight anti-parallel strands and two α-helices. The BH3 domain extends across 15 residues at the end of the second helix and eight amino acids in the chain following it. There are important structural differences in the BH3 domain in the intact SOUL molecule and the same sequence bound to Bcl-xL. PMID:21639858

A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor

PubMed Central

McEachern, Donna; Yang, Chao-Yie; Meagher, Jennifer; Stuckey, Jeanne; Wang, Shaomeng

2013-01-01

Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor growth but failed to achieve complete regression in vivo. We have now performed extensive modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-1074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with Ki values of < 1 nM and inhibits cancer cell growth with IC50 values of 1-2 nM in four small-cell lung cancer cell lines sensitive to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid, complete and durable tumor regression in vivo at a well-tolerated dose-schedule. Compound 32 is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date. PMID:23448298

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

PubMed Central

Murakawa, Tomokazu; Yamaguchi, Osamu; Hashimoto, Ayako; Hikoso, Shungo; Takeda, Toshihiro; Oka, Takafumi; Yasui, Hiroki; Ueda, Hiromichi; Akazawa, Yasuhiro; Nakayama, Hiroyuki; Taneike, Manabu; Misaka, Tomofumi; Omiya, Shigemiki; Shah, Ajay M.; Yamamoto, Akitsugu; Nishida, Kazuhiko; Ohsumi, Yoshinori; Okamoto, Koji; Sakata, Yasushi; Otsu, Kinya

2015-01-01

Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells. PMID:26146385

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival.

PubMed

Eichhorn, Joshua M; Alford, Sarah E; Sakurikar, Nandini; Chambers, Timothy C

2014-04-01

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status

PubMed Central

Mirjolet, J-F; Barberi-Heyob, M; Didelot, C; Peyrat, J-P; Abecassis, J; Millon, R; Merlin, J-L

2000-01-01

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC 50 values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity. © 2000 Cancer Research Campaign PMID:11044365

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

PubMed

Jiang, Tianyu; Liu, Minhao; Wu, Jianping; Shi, Yigong

2016-02-23

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses.

PubMed

Karpel-Massler, Georg; Ishida, Chiaki Tsuge; Bianchetti, Elena; Shu, Chang; Perez-Lorenzo, Rolando; Horst, Basil; Banu, Matei; Roth, Kevin A; Bruce, Jeffrey N; Canoll, Peter; Altieri, Dario C; Siegelin, Markus D

2017-07-01

Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors in vitro and in vivo in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. Cancer Res; 77(13); 3513-26. ©2017 AACR . ©2017 American Association for Cancer Research.

Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

PubMed Central

Guillemin, Yannis; Lopez, Jonathan; Gimenez, Diana; Fuertes, Gustavo; Valero, Juan Garcia; Blum, Loïc; Gonzalo, Philippe; Salgado, Jesùs; Girard-Egrot, Agnès; Aouacheria, Abdel

2010-01-01

Background The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. Methodology/Principal Findings Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. Conclusion/Significance BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains. PMID:20140092

Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.

PubMed

Yu, Albert Cheung Hoi; Yung, Hon Wa; Hui, Michael Hung Kit; Lau, Lok Ting; Chen, Xiao Qian; Collins, Richard A

2003-10-15

An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis. Copyright 2003 Wiley-Liss, Inc.

Natural Diterpenoid Compound Elevates Expression of Bim Protein, Which Interacts with Antiapoptotic Protein Bcl-2, Converting It to Proapoptotic Bax-like Molecule*

PubMed Central

Zhao, Lixia; He, Feng; Liu, Haiyang; Zhu, Yushan; Tian, Weili; Gao, Ping; He, Hongping; Yue, Wen; Lei, Xiaobo; Ni, Biyun; Wang, Xiaohui; Jin, Haijing; Hao, Xiaojiang; Lin, Jialing; Chen, Quan

2012-01-01

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics. PMID:22065578

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

PubMed

Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

2014-03-15

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function

PubMed Central

Holmes, Derek A; Yeh, Jung-Hua; Yan, Donghong; Xu, Min; Chan, Andrew C

2015-01-01

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival. PMID:25398911

Canonical Bcl-2 motifs of the Na+/K+ pump revealed by the BH3 mimetic chelerythrine: early signal transducers of apoptosis?

PubMed

Lauf, Peter K; Heiny, Judith; Meller, Jarek; Lepera, Michael A; Koikov, Leonid; Alter, Gerald M; Brown, Thomas L; Adragna, Norma C

2013-01-01

Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. CET inhibited NKP and NKCC by >90% (IC50 values ~35 and ~15 μM, respectively) without significant KCC activity change, and stimulated K+ loss by ~35% at 10-30 μM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET

Analysis of bax protein in sphingosine-induced apoptosis in the human leukemic cell line TF1 and its bcl-2 transfectants.

PubMed

Isogai, C; Murate, T; Tamiya-Koizumi, K; Yoshida, S; Ito, T; Nagai, H; Kinoshita, T; Kagami, Y; Hotta, T; Hamaguchi, M; Saito, H

1998-11-01

Sphingosine, a sphingolipid breakdown product, has been proposed as an apoptosis-inducing agent. In this study, we examined the effect of sphingosine in bcl-2-overexpressing cells compared with cells that do not express the bcl-2 gene. The human erythroleukemic cell line TF1, which lacks bcl-2 expression, was easily induced to undergo apoptotic cell death by a variety of stimuli, including depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) or exposure to methylmethane sulfonate (MMS) (100 microg/mL), ultraviolet light (15 J/m2), X-ray irradiation (20 Gy), or sphingosine, a sphingolipid breakdown product (5 microM). In contrast, bcl-2 transfectants of TF1 (TF1-bcl2), which we established, were resistant to most of these treatments but remained sensitive to sphingosine. Neither C2- nor C6-ceramide (short-chain ceramide) induced apoptosis in TF1-mock and TF1-bcl2 cells. Sphingosine-induced apoptosis could not be inhibited by fumonisin B1, which can prevent conversion of sphingosine to ceramide, suggesting that sphingosine itself, not ceramide, possesses apoptosis-inducing capability. Western blotting, which revealed a 21-kDa bax protein in untreated cells, revealed the presence of an additional 18-kDa protein in GM-CSF-depleted and MMS- or sphingosine-treated TF1-mock cells. In TF1-bcl2 cells, this protein was not detected after GM-CSF depletion or MMS treatment, but was observed after sphingosine treatment. Immunoprecipitation with anti-bcl2 antibody, followed by immunoblotting with anti-bax antibody, showed that both the 21-kDa bax protein and the 18-kDa protein heterodimerized with bcl-2 protein. These results suggest that sphingosine is a unique reagent for apoptosis and that it can overcome bcl-2 gene expression. Furthermore, induction of 18-kDa bax-related protein may play an important role in apoptosis. Sphingosine, but not ceramide, may prove applicable as a reagent for future cytotoxic drugs used to treat intractable tumors overexpressing

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

PubMed Central

Mahajan, Indra M.; Chen, Miao-Der; Muro, Israel; Robertson, John D.; Wright, Casey W.; Bratton, Shawn B.

2014-01-01

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax−/−Bak−/− cells and better than either Bid−/− or dominant-negative caspase-9-expressing cells. Only Bim−/− and Bax−/−Bak−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid−/− cells, it readily did so in Bim−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-XL with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members. PMID:24427286

Dual expression of MYC and BCL2 proteins predicts worse outcomes in diffuse large B-cell lymphoma.

PubMed

Clark Schneider, Kelli M; Banks, Peter M; Collie, Angela M B; Lanigan, Christopher P; Manilich, Elena; Durkin, Lisa M; Hill, Brian T; Hsi, Eric D

2016-07-01

Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials.

Hemagglutinating virus of Japan-artificial viral envelope liposome-mediated cotransfer of bag-1 and bcl-2 genes protects hepatic cells against ischemic injury through BAG-1-assisted preferential enhancement of bcl-2 protein expression.

PubMed

Yanada, Shinobu; Sasaki, Masao; Takayama, Shinichi; Kaneda, Yasufumi; Miwa, Nobuhiko

2005-05-01

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com; Cheah, Yew-Hoong; Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur

Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action ofmore » xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.« less

N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

PubMed

Zhao, Guoping; Neely, Aaron M; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G; Stivers, Nicole S; Burlison, Joseph A; White, Carl; Machen, Terry E; Li, Chi

2016-02-02

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.

N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

PubMed Central

Zhao, Guoping; Neely, Aaron M.; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G.; Stivers, Nicole S.; Burlison, Joseph A.; White, Carl; Machen, Terry E.; Li, Chi

2016-01-01

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok.

PubMed

D'Orsi, Beatrice; Mateyka, Julia; Prehn, Jochen H M

2017-10-01

Neuronal cell death is often triggered by events that involve intracellular increases in Ca 2+ . Under resting conditions, the intracellular Ca 2+ concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca 2+ overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca 2+ -dependent cell death. Excessive Ca 2+ entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer's disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca 2+ homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca 2+ -dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca 2+ homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the 'pro-apoptotic' Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such 'non-apoptotic/daytime' functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2.

PubMed

Renault, Thibaud T; Dejean, Laurent M; Manon, Stéphen

2017-01-01

Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells.

PubMed

Booth, Laurence; Roberts, Jane L; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Poklepovic, Andrew; Dent, Paul

2018-03-04

The irreversible ERBB1/2/4 inhibitor, neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of neratinib to promote greater levels of cell killing.

Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation.

PubMed

Fang, Difeng; Cui, Kairong; Hu, Gangqing; Gurram, Rama Krishna; Zhong, Chao; Oler, Andrew J; Yagi, Ryoji; Zhao, Ming; Sharma, Suveena; Liu, Pentao; Sun, Bing; Zhao, Keji; Zhu, Jinfang

2018-05-07

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b -deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

[Apoptosis-modulating effects of heat shock proteins: the influence of Hsp27 chaperone on TBA Bcl-2 family proteins in Jurkat cell line].

PubMed

Riazantseva, N V; Kaĭgorodova, E V; Maroshkina, A N; Belkina, M V; Novitskiĭ, V V

2012-01-01

The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers.

PubMed Central

Krajewska, M.; Krajewski, S.; Epstein, J. I.; Shabaik, A.; Sauvageot, J.; Song, K.; Kitada, S.; Reed, J. C.

1996-01-01

Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis-regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X-immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed

Cisplatin induces expression of drug resistance-related genes through c-jun N-terminal kinase pathway in human lung cancer cells.

PubMed

Xu, Li; Fu, Yingya; Li, Youlun; Han, Xiaoli

2017-08-01

Change of multidrug resistance-related genes (e.g., lung resistance protein, LRP) and overexpression of anti-apoptotic genes (Bcl-2, Bcl-Xl, XIAP, Survivin) are responsible for cisplatin resistance. In our study, we investigated the mechanism by which cisplatin induces LRP, Bcl-2, Bcl-xL, XIAP, and Survivin expression in human lung adenocarcinoma A549 cells and human H446 small cell lung cancer cells at mRNA and protein levels. In our study, cell proliferation was assessed with CCK-8 assays, and cell apoptosis was assessed with flow cytometric analysis and Annexin-V/PI staining. qPCR was used to complete RNA experiments. Protein expression was assessed with Western blotting. Cisplatin increased Bcl-2, LRP, and Survivin expression, but decreased Bcl-xL and XIAP expression in a dose-dependent manner. Preincubation with JNK-specific inhibitor, SP600125, significantly inhibited these genes' expression at mRNA and protein levels, enhanced chemosensitivity of lung cancer cells to cisplatin, and promoted cisplatin-induced apoptosis. Our data suggest that the JNK signaling pathway plays an important role in cisplatin resistance. Lung resistance protein (LRP) and anti-apoptotic genes (Bcl-2, Bcl-Xl, XIAP, Survivin) are involved in the process. The results reminded us of a novel therapy target for lung cancer treatment.

BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) not always convinces BAX (BCL-2-associated X protein) for apoptosis.

PubMed

Verma, Sharad; Goyal, Sukriti; Tyagi, Chetna; Jamal, Salma; Singh, Aditi; Grover, Abhinav

2016-06-01

The interaction of BAX (BCL-2-associated X protein) with BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) directly initiates BAX-mediated mitochondrial apoptosis. This molecular dynamics study reveals that BIM SAHB forms a stable complex with BAX but it remains in a non-functional conformation. N terminal of BAX folds towards the core which has been reported exposed in the functional monomer. The α1-α2 loop, which has been reported in open conformation in functional BAX, acquires a closed conformation during the simulation. BH3/α2 remains less exposed as compared to initial structure. The hydrophobic residues of BIM accommodates in the rear pocket of BAX during the simulation. A steep decrease in radius of gyration and solvent accessible surface area (SASA) indicates the complex folding to acquire a more stable but inactive conformation. Further the covariance matrix reveals that the backbone atoms' motions favour the inactive conformation of the complex. This is the first report on the non-functional BAX-BIM SAHB complex by molecular dynamics simulation in the best of our knowledge. Copyright © 2016 Elsevier Inc. All rights reserved.

Participation of GATA-3 in regulation of bone healing through transcriptional upregulation of bcl-xL expression

PubMed Central

Liao, Mei-Hsiu; Lin, Pei-I; Ho, Wei-Pin; Chan, Wing P; Chen, Ta-Liang; Chen, Ruei-Ming

2017-01-01

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-xL gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-xL gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures. PMID:29170477

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

PubMed

Zhang, X; Xu, Q; Saiki, I

2000-01-01

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

Immunohistochemical status of p53, MDM2, bcl2, bax, and ER in invasive ductal breast carcinoma in Tunisian patients.

PubMed

Baccouche, Sami; Daoud, Jamel; Frikha, Mounir; Mokdad-Gargouri, Raja; Gargouri, Ali; Jlidi, Rachid

2003-12-01

TP53 gene alterations have been associated with sporadic breast cancer. To assess the role of p53 in invasive ductal carcinoma (IDC) of the breast among Tunisian patients, p53 protein status was studied by immuno-histochemical analysis. The p53 protein was expressed in 41 of 70 (58%) tumors. Study of the status of its target gene expression showed that MDM2 was overexpressed in 43 tumors (61%), bcl2 in 29 (41%), and bax in only 9 (12%). Estrogen receptor (ER) was detected in 38 tumor tissues (54%). The accumulated p53 was significantly associated with MDM2-positive, bcl2-negative, and ER-negative tumors (P = 0.024, P = 0.000027, and P = 0.000008, respectively), whereas with bax the correlaton was not significant. Bcl2 immunostaining displayed a positive correlation with ER (P = 0.001). A significantly higher fraction of p53-positive cells was observed in ER-negative SBRII-SBRIII tumors than in ER-positive SBRI-SBRII tumors (P = 0.000066). bcl2-positive tumors were significantly correlated with ER-positive/SBRI-SBRII tumors (P = 0.007), but negatively correlated with p53/bax (P = 0000004). MDM2 immunostaining displayed the same phenotype as p53 in the correlation with bcl2 and ER (P = 0.003), strengthened by significant associations between MDM2-positive/p53-positive and bcl2-negative or ER-negative, respectively (P = 0.00005 and P = 0.000001, respectively). MDM2-positive cells were significantly correlated with the p53-positive/bax-negative phenotype (P = 0.04). These results suggest that p53 accumulated in these tumor tissues is associated with bad prognostic markers (ER-negative, SBRIII) of IDC. MDM2 overexpression might be responsible for the accumulated p53 value in IDC. Regulation of the apoptotic process is involved in IDC; bcl2 is associated with a good prognostic marker (ER-positive and SBRI-II), whereas the regulation of bax is complex and does not necessarily correlate with the overexpression of p53.

SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

PubMed

Jiang, Junjun; Li, Liyuan; Xie, Mingchao; Fuji, Ryosuke; Liu, Shangfeng; Yin, Xiaobei; Li, Genlin; Wang, Zhao

2015-01-01

Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples.

PubMed

Simons, Peter C; Young, Susan M; Carter, Mark B; Waller, Anna; Zhai, Dayong; Reed, John C; Edwards, Bruce S; Sklar, Larry A

2011-06-09

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast.

PubMed

Juhásová, Barbora; Mentel, Marek; Bhatia-Kiššová, Ingrid; Zeman, Igor; Kolarov, Jordan; Forte, Michael; Polčic, Peter

2011-09-02

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Therapeutics targeting Bcl-2 in hematological malignancies.

PubMed

Ruefli-Brasse, Astrid; Reed, John C

2017-10-23

Members of the B-cell lymphoma 2 ( BCL-2 ) gene family are attractive targets for cancer therapy as they play a key role in promoting cell survival, a long-since established hallmark of cancer. Clinical utility for selective inhibition of specific anti-apoptotic Bcl-2 family proteins has recently been realized with the Food and Drug Administration (FDA) approval of venetoclax (formerly ABT-199/GDC-0199) in relapsed chronic lymphocytic leukemia (CLL) with 17p deletion. Despite the impressive monotherapy activity in CLL, such responses have rarely been observed in other B-cell malignancies, and preclinical data suggest that combination therapies will be needed in other indications. Additional selective antagonists of Bcl-2 family members, including Bcl-X L and Mcl-1, are in various stages of preclinical and clinical development and hold the promise of extending clinical utility beyond CLL and overcoming resistance to venetoclax. In addition to direct targeting of Bcl-2 family proteins with BH3 mimetics, combination therapies that aim at down-regulating expression of anti-apoptotic BCL-2 family members or restoring expression of pro-apoptotic BH3 family proteins may provide a means to deepen responses to venetoclax and extend the utility to additional indications. Here, we review recent progress in direct and selective targeting of Bcl-2 family proteins for cancer therapy and the search for rationale combinations. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström Macroglobulinemia

PubMed Central

Gaudette, Brian T.; Dwivedi, Bhakti; Chitta, Kasyapa S.; Poulain, Stéphanie; Powell, Doris; Vertino, Paula; Leleu, Xavier; Lonial, Sagar; Chanan-Khan, Asher A.; Kowalski, Jeanne; Boise, Lawrence H.

2015-01-01

Waldenström Macroglobulinemia (WM) is a proliferative disorder of IgM secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing. PMID:25893290

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

PubMed

Grabarczyk, Piotr; Winkler, Passorn; Delin, Martin; Sappa, Praveen K; Bekeschus, Sander; Hildebrandt, Petra; Przybylski, Grzegorz K; Völker, Uwe; Hammer, Elke; Schmidt, Christian A

2018-03-01

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization. Copyright © 2018 American Society for Microbiology.

A novel histone deacetylase inhibitor, CG200745, potentiates anticancer effect of docetaxel in prostate cancer via decreasing Mcl-1 and Bcl-XL.

PubMed

Hwang, Jung Jin; Kim, Yong Sook; Kim, Taelim; Kim, Mi Joung; Jeong, In Gab; Lee, Je-Hwan; Choi, Jene; Jang, Sejin; Ro, Seonggu; Kim, Choung-Soo

2012-08-01

We synthesized a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), CG200745 {(E)-2-(Naphthalen-1-yloxymethyl)-oct-2-enedioic acid 1-[(3-dimethylamino-propyl)-amide] 8-hydroxyamide]}. Like other inhibitors, for example vorinostat and belinostat, CG200745 has the hydroxamic acid moiety to bind zinc at the bottom of catalytic pocket. Firstly, we analyzed its inhibitory activity against histone deacetylase (HDAC) in hormone-dependent LNCaP cells and hormone-independent DU145 and PC3 cells. CG200745 inhibited deacetylation of histone H3 and tubulin as much as vorinostat and belinostat did. CG200745 also inhibited growth of prostate cancer cells, increased sub-G1 population, and activated caspase-9, -3 and -8 in LNCaP, DU145 and PC3 cells. These results indicate that CG200745 induces apoptosis. Next, we examined the effect of CG200745 on cell death induced by docetaxel in DU145 cells in vitro and in vivo. Compared to mono-treatment with each drug, pre-treatment of DU145 cells with docetaxel followed by CG200745 showed synergistic cytotoxicity, and increased the apoptotic sub-G1 population, caspase activation, and tubulin acetylation. Moreover, the combination treatment decreased Mcl-1 and Bcl-(XL). Docetaxel and CG200745 combination reduced tumor size in the DU145 xenograft model. These preclinical results show that combination treatment with docetaxel and new HDACI, CG200745, potentiated anti-tumor effect in hormone-refractory prostate cancer (HRPC) cells via activation of apoptosis.

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF*

PubMed Central

Kilbride, Seán M.; Farrelly, Angela M.; Bonner, Caroline; Ward, Manus W.; Nyhan, Kristine C.; Concannon, Caoimhín G.; Wollheim, Claes B.; Byrne, Maria M.; Prehn, Jochen H. M.

2010-01-01

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation. PMID:20841353

Expression of c-Jun and Bcl-2 family proteins in apoptotic photoreceptors of RCS rats.

PubMed

Katai, Naomichi; Yanagidaira, Tomoko; Senda, Nami; Murata, Toshinori; Yoshimura, Nagahisa

2006-01-01

To determine if c-Jun and Bcl-2 family proteins play a role in photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. RCS and Sprague-Dawley rats were used. Cryosections of retinas harvested at various postnatal periods were immunostained with antibodies against c-Jun, Bcl-2, and Bax. Double staining with TdT-dUTP nick-end labeling (TUNEL) or propidium iodide (PI) and antibodies was also done. To study the time course of gene and protein expression, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting analyses were carried out. TUNEL-positive photoreceptors of RCS rats were stained strongly with antibodies against c-Jun and Bax. The number of immunoreactive cells increased on days 21 and 28 after birth (P21 and P28) and decreased on P45. Semiquantitative RT-PCR analysis showed that mRNAs for c-Jun and Bax were upregulated at P21 and P28, but those for Bcl-2 were unchanged. On immunoblotting, a 43-kDa band was revealed by the anti-c-Jun antibody and a 21-kDa band, by the anti-Bax antibody. Protein expression of c-Jun and Bax were increased at both P21 and P28. The temporal profiles of immunoreactivity, protein expression, and mRNA expression were similar. c-Jun and Bax may play a role in photoreceptor apoptosis in RCS rats.

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul

Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is knownmore » to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.« less

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose.

PubMed

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H M

2018-05-25

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose

PubMed Central

Lucantoni, Federico; Düssmann, Heiko; Llorente-Folch, Irene; Prehn, Jochen H.M.

2018-01-01

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors. PMID:29899841

Getting away with murder: how do the BCL-2 family of proteins kill with immunity?

PubMed Central

Renault, Thibaud T.; Chipuk, Jerry E.

2013-01-01

About 1 million per second is the number of white blood cells the adult human body produces. However, only a small fraction of them will survive as the majority is eliminated through a genetically controlled form of cell death referred to as apoptosis. This review places into perspective recent studies pertaining to the BCL-2 family of proteins as critical regulators of the development and function of the immune system, with particular attention on B cell and T cell biology. Here we discuss how elegant murine model systems have revealed the major contributions of the BCL-2 family in establishing an effective immune system. Moreover, we highlight some key regulatory pathways that influence the expression, function, and stability of individual BCL-2 family members, and discuss their role in immunity. From deadly methods to more gentle manners, the final portion of the review discusses the non-apoptotic functions of the BCL-2 family and how they pertain to the control of immunity. PMID:23527542

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

PubMed Central

Zekavati, Anna; Nasir, Asghar; Alcaraz, Amor; Aldrovandi, Maceler; Marsh, Phil; Norton, John D.; Murphy, John J.

2014-01-01

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in

The Impact of Exercise on the Vulnerability of Dopamine Neurons to Cell Death in Animal Models of Parkinson’s Disease

DTIC Science & Technology

2008-07-01

increase in the apparent activity of MEK1/ 2 and a decrease in the apparent activity of protein phosphatase 2A. Third, the pro-survival protein Bcl - 2 ...2000. Correlation between structure of Bcl - 2 and its inhibitory function of JNK and caspase activity in dopa- minergic neuronal apoptosis. J Neurochem 74...Choi WS, Kim JE, Seo JW, O’Malley KL, Oh YJ. 1998. Over- expression of HA-Bax but not Bcl - 2 or Bcl -XL attenuates 6-hydroxy- dopamine-induced neuronal

Akt regulates drug-induced cell death through Bcl-w downregulation.

PubMed

Garofalo, Michela; Quintavalle, Cristina; Zanca, Ciro; De Rienzo, Assunta; Romano, Giulia; Acunzo, Mario; Puca, Loredana; Incoronato, Mariarosaria; Croce, Carlo M; Condorelli, Gerolama

2008-01-01

Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with pro-apoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w.

Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

PubMed

Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

2015-02-14

To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P < 0.05). A cell proliferation assay revealed that B7-H3 overexpression increased the drug resistance of cells and resulted in a higher survival rate (P < 0.05). In addition, the results of cell cycle and active caspase-3 western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines (P < 0.05). B7-H3 overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P < 0.05). After treating B7-H3 overexpressing cells with the Jak2

p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway.

PubMed

Thomas, A; Giesler, T; White, E

2000-11-02

A member of the small G protein family, cdc42, was isolated from a screen undertaken to identify p53-inducible genes during apoptosis in primary baby rat kidney (BRK) cells transformed with E1A and a temperature-sensitive mutant p53 using a PCR-based subtractive hybridization method. Cdc42 is a GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. In response to external stimuli, Cdc42 is known to transduce signals to regulate the organization of the actin cytoskeleton, induce DNA synthesis in quiescent fibroblasts, and promote apoptosis in neuronal and immune cells. In this study, we have demonstrated that cdc42 mRNA and protein were up-regulated in the presence of wild-type p53 in BRK cells, followed by cytoplasmic to plasma membrane translocation of Cdc42. Overexpression of Cdc42 in the presence of a dominant-negative mutant p53 induced apoptosis rapidly, indicating that Cdc42 functions downstream of p53. Furthermore, stable expression of a dominant-negative mutant of Cdc42 partially inhibited p53-mediated apoptosis. The Bcl-2 family members Bcl-xL, and the adenovirus protein E1B 19K, inhibited Cdc42-mediated apoptosis, whereas Bcl-2 did not. We provide evidence that PAK1 and JNK1 may play a role downstream of Cdc42 to transduce its apoptotic signal. Cdc42/PAK1 activates JNK1-induced phosphorylation of Bcl-2, thereby inactivating its function, and that a phosphorylation resistant mutant (Bcl-2S70,87A,T56,74A) gains the ability to inhibit Cdc42- and p53-mediated apoptosis. Thus, one mechanism by which p53 promotes apoptosis is through activation of Cdc42 and inactivation of Bcl-2.

Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.

PubMed

Okamoto, Toru; Campbell, Stephanie; Mehta, Ninad; Thibault, John; Colman, Peter M; Barry, Michele; Huang, David C S; Kvansakul, Marc

2012-11-01

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.

Non-endometrioid and high-grade endometrioid endometrial cancers show DNA fragmentation factor 40 (DFF40) and B-cell lymphoma 2 protein (BCL2) underexpression, which predicts disease-free and overall survival, but not DNA fragmentation factor 45 (DFF45) underexpression.

PubMed

Banas, Tomasz; Pitynski, Kazimierz; Okon, Krzysztof; Winiarska, Aleksandra

2018-04-13

The expression of DNA fragmentation factor 45 (DFF45) and B-cell lymphoma 2 (BCL2) in glands of the normal human endometrium is related to phases of the menstrual cycle and decreases after menopause, whereas the expression of DNA fragmentation factor 40 (DFF40) is stable. Moreover, DF45, BCL2 and DFF40 underexpression has been reported in numerous malignancies, including uterine leiomyosarcomas. In this study, we aimed to investigate DFF45, BCL2 and DFF40 expression in endometrioid and non-endometrioid types of endometrial cancers (ECs). We also evaluated the correlations between DFF45, BCL2 and DFF40 expression levels and clinicopathological parameters and determined the value of these three proteins as prognostic markers of disease-free survival (DFS) and overall survival (OS). Immunohistochemistry was performed to evaluate DFF45, BCL2 and DFF40 expression in 342 cases of ECs. Student's t-test, the Mann-Whitney U-test, and the chi-squared test were used for the statistical analyses as appropriate. The Cox-Mantel test, Cox's proportional hazard model, and relative risk analyses were used to evaluate associations between DFF40, DFF45, and BCL2 expression and clinicopathological characteristics. DFF40 and BCL2, but not DFF45, were significantly underexpressed in non-endometrioid and high-grade endometrioid ECs compared with low- and moderate-grade endometrioid ECs. Women with DFF40- and BCL2-negative tumors had higher risks of disease recurrence, lymph node involvement, lympho-vascular space infiltration, and deep myometrial invasion compared with women with DFF40- and BCL2-positive tumors. Additionally, women with DFF40- and BCL2-negative tumors had significantly lower OS and DFS than women with DFF40- and BCL2-positive tumors. A multivariable analysis of the model, including the clinicopathological characteristics and immunohistochemical results, showed that negative BCL2 expression, lymph node involvement, and high-stage and high-grade disease were independent

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

PubMed

Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

2014-09-01

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium.

PubMed

Kamdar, O; Le, Wei; Zhang, J; Ghio, A J; Rosen, G D; Upadhyay, D

2008-10-29

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jauharoh, Siti Nur Aisyah; Faculty of Medicine and Health Science, Syarif Hidayatullah State Islamic University, Jakarta 15412; Saegusa, Jun

Highlights: Black-Right-Pointing-Pointer Ro52{sup low} HeLa cells are resistant to apoptosis upon various stimulations. Black-Right-Pointing-Pointer Ro52 is upregulated by IFN-{alpha}, etoposide, or IFN-{gamma} and anti-Fas Ab. Black-Right-Pointing-Pointer Ro52-mediated apoptosis is independent of p53. Black-Right-Pointing-Pointer Ro52 selectively regulates Bcl-2 expression. -- Abstract: SS-A/Ro52 (Ro52), an autoantigen in systemic autoimmune diseases such as systemic lupus erythematosus and Sjoegren's syndrome, has E3 ligase activity to ubiquitinate proteins that protect against viral infection. To investigate Ro52's role during stress, we transiently knocked it down in HeLa cells by siRo52 transfection. We found that Ro52{sup low} HeLa cells were significantly more resistant to apoptosis than wild-typemore » HeLa cells when stimulated by H{sub 2}O{sub 2}- or diamide-induced oxidative stress, IFN-{alpha}, IFN-{gamma} and anti-Fas antibody, etoposide, or {gamma}-irradiation. Furthermore, Ro52-mediated apoptosis was not influenced by p53 protein level in HeLa cells. Depleting Ro52 in HeLa cells caused Bcl-2, but not other Bcl-2 family molecules, to be upregulated. Taken together, our data showed that Ro52 is a universal proapoptotic molecule, and that its proapoptotic effect does not depend on p53, but is exerted through negative regulation of the anti-apoptotic protein Bcl-2. These findings shed light on a new physiological role for Ro52 that is important to intracellular immunity.« less

MeHG Stimulates Antiapoptotic Signaling in Stem Cells

DTIC Science & Technology

2010-07-01

Soane, L., Siegel, Z. T., Schuh, R. A. & Fiskum, G. (2008). Postnatal developmental regulation of Bcl - 2 family proteins in brain mitochondria. J...resulting in neutralization of the anti-apoptotic members Bcl -xL and Bcl - 2 [26]. In neurons, the absence or inhibition of p53 activity protects...Tiwari M and Godbole MM (2003) Hypothyroidism alters the expression of Bcl - 2 family genes to induce enhanced apoptosis in the developing

Bcl-2 antisense therapy in B-cell malignancies.

PubMed

Chanan-Khan, Asher

2005-07-01

Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

PubMed Central

Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

2016-01-01

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

Ultra High Throughput Screening of Natural Product Extracts to Identify Pro-apoptotic Inhibitors of Bcl-2 Family Proteins

PubMed Central

Hassig, Christian A.; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W.; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J.; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E.; Brown, Susan G.; Baire, Beeraiah; Michel, Andrew R.; Hoye, Thomas R.; Francis, Subhashree; Georg, Gunda I.; Walters, Michael A.; Divlianska, Daniela B.; Roth, Gregory P.; Wright, Amy E.; Reed, John C.

2015-01-01

Anti-apoptotic Bcl-2 family proteins are validated cancer targets comprised of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). While several isoform-selective inhibitors have been developed using structure-based design or high throughput screening (HTS) of synthetic chemical libraries, no large scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six anti-apoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally-relevant PPIs. The screens were conducted in 1,536-well format and displayed satisfactory overall HTS statistics, with Z’-factor values ranging from 0.72 to 0.83, and a hit confirmation rate between 16-64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra high throughput screening using natural product sources and highlight some of the challenges associated with this approach. PMID:24870016

A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo

PubMed Central

Petros, Andrew M; Nettesheim, David; van Dyk, Linda F.; Labrada, Lucia; Speck, Samuel H; Levine, Beth

2005-01-01

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the γHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type γHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of γHV68 from latency and efficient persistent γHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection. PMID:16201011

Design of Bcl-2 and Bcl-xL Inhibitors with Subnanomolar Binding Affinities Based upon a New Scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Haibin; Chen, Jianfang; Meagher, Jennifer L.

Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal structure of Bcl-xL and U.S. Food and Drug Administration (FDA) approved drugs and was found to have an affinity of 100 {micro}M for both Bcl-2 and Bcl-xL. Linking this weak lead to another weak-affinity fragment derived from Abbott's ABT-737 led to an improvement of the binding affinity by a factor of >10,000. Further optimization ultimately yielded compounds with subnanomolar binding affinities for both Bcl-2 andmore » Bcl-xL and potent cellular activity. The best compound (21) binds to Bcl-xL and Bcl-2 with K{sub i} < 1 nM, inhibits cell growth in the H146 and H1417 small-cell lung cancer cell lines with IC{sub 50} values of 60-90 nM, and induces robust cell death in the H146 cancer cell line at 30-100 nM.« less

A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells

PubMed Central

Shen, Betty W.; Song, Yifan; Frayo, Shani; Convertine, Anthony J.; Margineantu, Daciana; Booth, Garrett; Correia, Bruno E.; Cheng, Yuanhua; Schief, William R.; Hockenbery, David M.; Press, Oliver W.; Stoddard, Barry L.; Stayton, Patrick S.; Baker, David

2014-01-01

SUMMARY Since apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homologue of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions, but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High specificity designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates. PMID:24949974

Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53

PubMed Central

Banu, Sakhila K.; Stanley, Jone A.; Lee, JeHoon; Stephen, Sam D.; Arosh, Joe A.; Hoyer, Patricia B.; Burghardt, Robert C.

2011-01-01

Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells were treated with 10 μM potassium dichromate (CrVI) for 12 and 24 h, with or without vitamin C pre-treatment for 24 h. The effects of CrVI on intrinsic apoptotic pathway(s) were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C. PMID:21262251

Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banu, Sakhila K., E-mail: skbanu@cvm.tamu.edu; Stanley, Jone A.; Lee, JeHoon

Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells were treated with 10 {mu}M potassium dichromate (CrVI) for 12 and 24 h, with or without vitamin C pre-treatment for 24 h. The effects of CrVI on intrinsic apoptotic pathway(s)more » were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C.« less

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Persea declinata (Bl.) Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

PubMed Central

Wong, Yi Li; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A. Hamid A.

2014-01-01

Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

Segmental heterogeneity in Bcl-2, Bcl-xL and Bax expression in rat tubular epithelium after ischemia-reperfusion.

PubMed

Valdés, Francisco; Pásaro, Eduardo; Díaz, Inmaculada; Centeno, Alberto; López, Eduardo; García-Doval, Sandra; González-Roces, Severino; Alba, Alfonso; Laffon, Blanca

2008-06-01

Studies in rats with bilateral clamping of renal arteries showed transient Bcl-2, Bcl-xL and Bax expression in renal tubular epithelium following ischemia-reperfusion. However, current data on the preferential localization of specific mRNAs or proteins are limited because gene expression was not analysed at segmental level. This study analyses the mRNA expression of Bcl-2, Bcl-xL and Bax in four segments of proximal and distal tubules localized in the renal cortex and outer medulla in rat kidneys with bilateral renal clamping for 30 min and seven reperfusion times versus control animals without clamp. Proximal convoluted tubule (PCT), distal convoluted tubule (DCT), proximal straight tubule (PST) and medullary thick ascending limb (MTAL) were obtained by manual microdissection. RT-PCR was used to analyse mRNA expression at segmental level. Proximal convoluted tubule and MTAL showed early, persistent and balanced up-regulation of Bcl-2, Bcl-xL and Bax, while PST and DCT revealed only Bcl-2 and Bcl-xL, when only Bax was detected in PST. DCT expressed Bcl-xL initially, and persistent Bcl-2 later. These patterns suggest a heterogeneous apoptosis regulatory response in rat renal tubules after ischemia-reperfusion, independently of cortical or medullary location. This heterogeneity of the expression patterns of Bcl-2 genes could explain the different susceptibility to undergo apoptosis, the different threshold to ischemic damage and the different adaptive capacity to injury among these tubular segments.

The Bcl-2 apoptotic switch in cancer development and therapy

PubMed Central

Adams, JM; Cory, S

2009-01-01

Impaired apoptosis is both critical in cancer development and a major barrier to effective treatment. In response to diverse intracellular damage signals, including those evoked by cancer therapy, the cell’s decision to undergo apoptosis is determined by interactions between three factions of the Bcl-2 protein family. The damage signals are transduced by the diverse ‘BH3-only’ proteins, distinguished by the BH3 domain used to engage their pro-survival relatives: Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1. This interaction ablates pro-survival function and allows activation of Bax and Bak, which commit the cell to apoptosis by permeabilizing the outer membrane of the mitochondrion. Certain BH3-only proteins (e.g. Bim, Puma) can engage all the pro-survival proteins, but others (e.g. Bad, Noxa) engage only subsets. Activation of Bax and Bak appears to require that the BH3-only proteins engage the multiple pro-survival proteins guarding Bax and Bak, rather than binding to the latter. The balance between the pro-survival proteins and their BH3 ligands regulates tissue homeostasis, and either overexpression of a pro-survival family member or loss of a proapoptotic relative can be oncogenic. Better understanding of the Bcl-2 family is clarifying its role in cancer development, revealing how conventional therapy works and stimulating the search for ‘BH3 mimetics’ as a novel class of anticancer drugs. PMID:17322918

NetpathXL - An Excel Interface to the Program NETPATH

USGS Publications Warehouse

Parkhurst, David L.; Charlton, Scott R.

2008-01-01

NetpathXL is a revised version of NETPATH that runs under Windows? operating systems. NETPATH is a computer program that uses inverse geochemical modeling techniques to calculate net geochemical reactions that can account for changes in water composition between initial and final evolutionary waters in hydrologic systems. The inverse models also can account for the isotopic composition of waters and can be used to estimate radiocarbon ages of dissolved carbon in ground water. NETPATH relies on an auxiliary, database program, DB, to enter the chemical analyses and to perform speciation calculations that define total concentrations of elements, charge balance, and redox state of aqueous solutions that are then used in inverse modeling. Instead of DB, NetpathXL relies on Microsoft Excel? to enter the chemical analyses. The speciation calculation formerly included in DB is implemented within the program NetpathXL. A program DBXL can be used to translate files from the old DB format (.lon files) to NetpathXL spreadsheets, or to create new NetpathXL spreadsheets. Once users have a NetpathXL spreadsheet with the proper format, new spreadsheets can be generated by copying or saving NetpathXL spreadsheets. In addition, DBXL can convert NetpathXL spreadsheets to PHREEQC input files. New capabilities in PHREEQC (version 2.15) allow solution compositions to be written to a .lon file, and inverse models developed in PHREEQC to be written as NetpathXL .pat and model files. NetpathXL can open NetpathXL spreadsheets, NETPATH-format path files (.pat files), and NetpathXL-format path files (.pat files). Once the speciation calculations have been performed on a spreadsheet file or a .pat file has been opened, the NetpathXL calculation engine is identical to the original NETPATH. Development of models and viewing results in NetpathXL rely on keyboard entry as in NETPATH.

Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

PubMed

Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

2015-07-16

p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

Predictive Bcl-2 Family Binding Models Rooted in Experiment or Structure

PubMed Central

DeBartolo, Joe; Dutta, Sanjib; Reich, Lothar; Keating, Amy E.

2013-01-01

Proteins of the Bcl-2 family either enhance or suppress programmed cell death and are centrally involved in cancer development and resistance to chemotherapy. BH3 (Bcl-2 homology 3)-only Bcl-2 proteins promote cell death by docking an α-helix into a hydrophobic groove on the surface of one or more of five pro-survival Bcl-2 receptor proteins. There is high structural homology within the pro-death and pro-survival families, yet a high degree of interaction specificity is nevertheless encoded, posing an interesting and important molecular recognition problem. Understanding protein features that dictate Bcl-2 interaction specificity is critical for designing peptide-based cancer therapeutics and diagnostics. In this study, we present peptide SPOT arrays and deep sequencing data from yeast display screening experiments that significantly expand the BH3 sequence space that has been experimentally tested for interaction with five human anti-apoptotic receptors. These data provide rich information about the determinants of Bcl-2 family specificity. To interpret and use the information, we constructed two simple data-based models that can predict affinity and specificity when evaluated on independent data sets within a limited sequence space. We also constructed a novel structure-based statistical potential, called STATIUM, which is remarkably good at predicting Bcl-2 affinity and specificity, especially considering it is not trained on experimental data. We compare the performance of our three models to each other and to alternative structure-based methods and discuss how such tools can guide prediction and design of new Bcl-2 family complexes. PMID:22617328

BCL-2: Long and winding path from discovery to therapeutic target

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schenk, Robyn L.; Strasser, Andreas; Department of Medical Biology, University of Melbourne, Parkville, Melbourne, Victoria 3010

In 1988, the BCL-2 protein was found to promote cancer by limiting cell death rather than enhancing proliferation. This discovery set the wheels in motion for an almost 30 year journey involving many international research teams that has recently culminated in the approval for a drug, ABT-199/venetoclax/Venclexta that targets this protein in the treatment of cancer. This review will describe the long and winding path from the discovery of this protein and understanding the fundamental process of apoptosis that BCL-2 and its numerous homologues control, through to its exploitation as a drug target that is set to have significant benefitmore » for cancer patients. - Highlights: • BCL-2 proteins control the intrinsic or mitochondrial pathway of apoptosis. • Defective apoptosis is a hallmark of cancer. • BH3-mimetics inhibit pro-survival BCL-2 proteins to induce cancer cell death. • ABT-199/venetoclax is approved for treatment of chronic lymphocytic leukaemia.« less

Structure based re-design of the binding specificity of anti-apoptotic Bcl-xL

PubMed Central

Chen, T. Scott; Palacios, Hector; Keating, Amy E.

2012-01-01

Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Re-designed proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to re-engineer the multi-specific anti-apoptotic protein Bcl-xL to remove its interactions with many of its binding partners, making it a high affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-xL sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-xL variants, we successfully identified a protein with ~1,000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best re-designed protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. PMID:23154169

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA

PubMed Central

Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K.; Kio, Ebenezer A.; Smith, James C.; Pineda-Roman, Mauricio; Stuart, Robert K.; Spicer, Eleanor K.

2007-01-01

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA–stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2 mRNA and protein but no change in β-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin. PMID:17179226

Similar BCL-X but different BCL-2 levels in the two age groups of north African nasopharyngeal carcinomas.

PubMed

Khabir, Abdelmajid; Ghorbel, Abdelmoneem; Daoud, Jamel; Frikha, Mounir; Drira, Mohamed Mokhtar; Laplanche, Agnès; Busson, Pierre; Jlidi, Rachid

2003-01-01

Nasopharyngeal carcinomas (NPCs) are consistently associated with the Epstein-Barr virus (EBV). As Bcl-2 and Bcl-X are co-expressed in EBV-transformed B-lymphocytes, we attempted to determine their status in malignant NPC cells. A retrospective series of 100 NPC specimens from untreated Tunisian patients was investigated by immuno-histochemistry. Twenty seven of the patients were below 30 years old and therefore classified in the "juvenile" form of north African NPCs. Bcl-2 and Bcl-X expression was assessed semi-quantitatively using a score based on the percentage of positive cells and staining intensity. Intense Bcl-X expression was detected in malignant cells of 100% biopsy samples with similar scores for patients below 30 years or those aged 30 or over. Bcl-2 was detected in 89% biopsies but its expression differed considerably between the samples. The average Bcl-2 score was much lower for patients under 30 years (4.4+/-1.5 compared to 6.5+/-2 for older patients; P<10(-6)). Multivariate analysis demonstrated that no other clinical parameter, except the primary tumor size, was correlated to the Bcl-2 score. Bcl-X and Bcl-2 are co-expressed in 89% of NPCs whereas their expression is mutually exclusive in other head and neck carcinomas (particularly squamous cell carcinomas, SCC). The constantly high expression of Bcl-X is consistent with it being induced by the EBV protein Epstein-Barr nuclear antigen 1 (EBNA1), as recently reported in a murine model. The contrasted levels of Bcl-2 expression in the two age groups strengthen the hypothesis that these clinical forms result from distinct oncogenic mechanisms.

BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints

PubMed Central

Fischer, Axel W.; Alexander, Nathan S.; Woetzel, Nils; Karakaş, Mert; Weiner, Brian E.; Meiler, Jens

2016-01-01

For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The protein-size-normalized root-mean-square-deviation (RMSD100) value of the most accurate model is better than 8 Å for twenty-seven, better than 6 Å for twenty-two, and better than 4 Å for fifteen out of twenty-nine proteins, demonstrating the algorithm’s ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data. PMID:25820805

Gossypol inhibits phosphorylation of Bcl-2 in human leukemia HL-60 cells.

PubMed

Huang, Li-heng; Hu, Jia-qi; Tao, Wei-qun; Li, Yuan-hong; Li, Guan-ming; Xie, Pei-yi; Liu, Xiao-shan; Jiang, Jikai

2010-10-25

Gossypol is an attractive therapeutic anti-tumor agent as an apoptosis inducer and is being evaluated in preclinical tests. However, the molecular mechanisms underlying apoptosis induction by gossypol in malignant cells have not been completely enunciated. Here we investigate the alterations of Bcl-2/Bcl-xL/Mcl-1 protein levels and Bcl-2 phosphorylation in gossypol-induced apoptosis in human leukemia HL-60 cells. We found that gossypol treatment inhibited cell growth and induced apoptosis in HL-60 cells. Bcl-2/Bcl-xL/Mcl-1 protein levels were slightly reduced and phosphorylation of Bcl-2 at threonine 56 (phospho T56) was not altered. However, phosphorylation of Bcl-2 at serine 70 (phospho S70) was strikingly down-regulated in gossypol-exposed cells. This reduction was found to be not only in both dose- and time-dependent fashion but also obviated by phorbol l2,13-dibutyrate (PDBu), an activator of protein kinase C (PKC). In addition, pre-treatment of PDBu partially prevented gossypol-induced apoptosis in HL-60 cells. Collectively, gossypol treatment can reduce phosphorylation of Bcl-2 at serine 70 in leukemia HL-60 cells and gossypol may be a promising therapeutical candidate for leukemia patients especially expressing phosphorylated Bcl-2 at Ser70. Copyright 2010 Elsevier B.V. All rights reserved.

Endogenous association of Bim BH3-only protein with Mcl-1, Bcl-xL and Bcl-2 on mitochondria in human B cells.

PubMed

Gomez-Bougie, Patricia; Bataille, Régis; Amiot, Martine

2005-03-01

Bim is an essential regulator of lymphoid system homeostasis and appears essential for B cell apoptosis induction. The mechanism by which Bim isoforms are held in an inactive form remains poorly documented in normal B cells. In the current study, we demonstrated that in normal tonsil B cells the three major Bim isoforms are strongly associated with the anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-x(L). On the other hand, only a weak association of BimEL and L with the dynein LC8 chain has been found. In addition, there is no free Bim in normal B cells. Moreover, subcellular fractionation demonstrated that Bim and the anti-apoptotic counterparts are localized preferentially in the mitochondria-rich fraction. The fact that most Bim was found in this fraction supports the hypothesis that it is sequestered by anti-apoptotic molecules in mitochondria where its pro-apoptotic activity is controlled. Of interest, BimS is essentially complexed to Mcl-1 and the Mcl-1/Bim complex is the most abundant among the three types of complexes. This supports the idea that this complex is critical for the control of B cell death. In conclusion, these results favor a model in which Bim release from anti-apoptotic proteins is a critical event for initiation of apoptosis.

ΔN-Bcl-xL, a therapeutic target for neuroprotection

PubMed Central

Park, Han-A; Jonas, Elizabeth A.

2017-01-01

The B-cell lymphoma-extra large (Bcl-xL) is a mitochondrial anti-apoptotic protein that plays a role in neuroprotection. However, during excitotoxic stimulation, Bcl-xL undergoes caspase-dependent cleavage and produces a fragmented form, ΔN-Bcl-xL. Accumulation of ΔN-Bcl-xL is associated with mitochondrial dysfunction and neuronal death. Therefore, strategies to inhibit the activity or formation of ΔN-Bcl-xL protect the brain against excitotoxic injuries. Our team found that the pharmacological inhibitor ABT-737 exerts dose dependent effects in primary neurons. When primary hippocampal neurons were treated with 1 μM ABT-737, glutamate-mediated mitochondrial damage and neuronal death were exacerbated, whereas 10 nM ABT-737, a 100-fold lower concentration, protected mitochondrial function and enhanced neuronal viability against glutamate toxicity. In addition, we suggested acute vs. prolonged formation of ΔN-Bcl-xL may have different effects on mitochondrial or neuronal functions. Unlike acute production of ΔN-Bcl-xL by glutamate, overexpression of ΔN-Bcl-xL did not cause drastic changes in neuronal viability. We predicted that neurons undergo adaptation and may activate altered metabolism to compensate for ΔN-Bcl-xL-mediated mitochondrial dysfunction. Although the detailed mechanism of ABT-mediated neurotoxicity neuroprotection is still unclear, our study shows that the mitochondrial membrane protein ΔN-Bcl-xL is a central target for interventions. PMID:29239317

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kappa B and inhibition of Bcl-xl expression.

PubMed

Huerta-Yepez, Sara; Vega, Mario; Jazirehi, Ali; Garban, Hermes; Hongo, Fumiya; Cheng, Genhong; Bonavida, Benjamin

2004-06-24

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues and this prompted its potential therapeutic application in cancer. However, not all cancers are sensitive to TRAIL-mediated apoptosis and, therefore, TRAIL-resistant cancer cells must be sensitized first to become sensitive to TRAIL. Treatment of prostate cancer (CaP) cell lines (DU145, PC-3, CL-1, and LNCaP) with nitric oxide donors (e.g. (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate)) sensitized CaP cells to TRAIL-induced apoptosis and synergy was achieved. The mechanism by which DETANONOate mediated the sensitization was examined. DETANONOate inhibited the constitutive NF-kappa B activity as assessed by EMSA. Also, p50 was S-nitrosylated by DETANONOate resulting in inhibition of NF-kappa B. Inhibition of NF-kappa B activity by the chemical inhibitor Bay 11-7085, like DETANONOate, sensitized CaP to TRAIL apoptosis. In addition, DETANONOate downregulated the expression of Bcl-2 related gene (Bcl-(xL)) which is under the transcriptional regulation of NF-kappa B. The regulation of NF-kappa B and Bcl-(xL) by DETANONOate was corroborated by the use of Bcl-(xL) and Bcl-x kappa B reporter systems. DETANONOate inhibited luciferase activity in the wild type and had no effect on the mutant cells. Inhibition of NF-kappa B resulted in downregulation of Bcl-(xL) expression and sensitized CaP to TRAIL-induced apoptosis. The role of Bcl-(xL) in the regulation of TRAIL apoptosis was corroborated by inhibiting Bcl-(xL) function by the chemical inhibitor 2-methoxyantimycin A(3) and this resulted in sensitization of the cells to TRAIL apoptosis. Signaling by DETANONOate and TRAIL for apoptosis was examined. DETANONOate altered the mitochondria by inducing membrane depolarization and releasing modest amounts of cytochrome c and Smac/DIABLO in the absence of

Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

PubMed

Park, Shin-Young; Ma, Weina; Yoon, Sung Nyo; Kang, Min Jeong; Han, Joong-Soo

2015-01-01

We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

Corrected and Republished from: BCL11A Is a Critical Component of a Transcriptional Network That Activates Recombinase Activating Gene Expression and V(D)J Recombination

PubMed Central

Lee, Baeck-Seung; Lee, Bum-Kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.

2017-01-01

ABSTRACT Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:29038163

mRNA in exosomas as a liquid biopsy in non-Hodgkin Lymphoma: a multicentric study by the Spanish Lymphoma Oncology Group.

PubMed

Provencio, Mariano; Rodríguez, Marta; Cantos, Blanca; Sabín, Pilar; Quero, Cristina; García-Arroyo, Francisco R; Rueda, Antonio; Maximiano, Constanza; Rodríguez-Abreu, Delvys; Sánchez, Antonio; Silva, Javier; García, Vanesa

2017-08-01

To determine the feasibility of mRNAs ( C-MYC, BCL-XL, BCL-6, NF-κβ, PTEN and AKT ) in exosomes of plasma as a liquid biopsy method for monitoring and prognostic evolution in B-cell lymphomas. Exosomes were isolated from 98 patients with B-cell Lymphoma and 68 healthy controls. mRNAs were analyzed by quantitative PCR. An additional 31 post-treatment samples were also studied. In the general and follicular lymphoma series, the presence of AKT mRNA was associated with poor response to rituximab-based treatment. Patients with first relapse or disease progression showed a lower percentage of PTEN and BCL-XL mRNA. The presence of BCL-6 mRNA was associated with a high death rate. The absence of PTEN mRNA in the general series, and presence of C-MYC mRNA in follicular lymphomas, were associated with short progression-free survival. BCL-6 and C-MYC mRNA were independent prognostic variables of overall survival. C-MYC mRNA may provide prognostic information with respect to overall survival. BCL-XL mRNA and increase of BCL-6 mRNA in post-treatment samples could serve as molecular monitoring markers. This is the first large study to evaluate the prognostic and predictive values of pretreatment tumor-associated mRNA in exosomes. BCL-6 and C-MYC mRNA positivity in pretreatment samples were predictors of worse PFS compared to patients with mRNA negativity. C-MYC mRNA positivity was also a statistically significant predictor of inability to obtain complete response with first-line therapy.

Pegasus XL CYGNSS Rollout

NASA Image and Video Library

2016-11-28

At Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is transported from the hangar at Building 1555 to be mated to L-1011 carrier aircraft near Vandenberg's runway. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Estradiol increases the Bax/Bcl-2 ratio and induces apoptosis in the anterior pituitary gland.

PubMed

Zaldivar, Verónica; Magri, María Laura; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Radl, Daniela; Ferraris, Jimena; Pisera, Daniel; Seilicovich, Adriana

2009-01-01

Estrogens are recognized as acting as modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals, thus participating in anterior pituitary homeostasis during the estrous cycle. The balance of pro- and antiapoptotic proteins of the Bcl-2 family is known to regulate cell survival and apoptosis. In order to understand the mechanisms underlying apoptosis during the estrous cycle, we evaluated the expression of the proapoptotic protein Bax and the antiapoptotic proteins Bcl-2 and Bcl-xL in the anterior pituitary gland in cycling female rats as well as the influence of estradiol on the expression of these proteins in anterior pituitary cells of ovariectomized rats. As determined by Western blot, the expression of Bax was higher in anterior pituitary glands from rats at proestrus than at diestrus I, Bcl-2 protein levels showed no difference and Bcl-xL expression was lower, thus increasing the Bax/Bcl-2 ratio at proestrus. Assessed by annexin V binding and flow cytometry, the percentage of apoptotic anterior pituitary cells was higher in rats at proestrus than at diestrus I. Chronic estrogen treatment in ovariectomized rats enhanced the Bax/Bcl-2 ratio and induced apoptosis. Moreover, incubation of cultured anterior pituitary cells from ovariectomized rats with 17beta-estradiol for 24 h increased the Bax/Bcl-2 ratio, decreased Bcl-xL expression and induced apoptosis. Our results demonstrate that estradiol increases the ratio between proapoptotic and antiapoptotic proteins of the Bcl-2 family. This effect could participate in the sensitizing action of estrogens to proapoptotic stimuli and therefore be involved in the high apoptotic rate observed at proestrus in the anterior pituitary gland.

FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor

NASA Astrophysics Data System (ADS)

Du, Mengyan; Yang, Fangfang; Mai, Zihao; Qu, Wenfeng; Lin, Fangrui; Wei, Lichun; Chen, Tongsheng

2018-04-01

We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.

uPAR and Cathepsin B Downregulation Induces Apoptosis by Targeting Calcineurin A to BAD via Bcl-2 in Glioma

PubMed Central

Malla, Rama Rao; Gopinath, Sreelatha; Gondi, Christopher S.; Alapati, Kiranmai; Dinh, Dzung H.; Tsung, Andrew J.; Rao, Jasti S.

2011-01-01

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ, and increased the interaction of BAD with Bcl-Xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD and caspase 3 as well as PARP activation, cytochrome c and SMAC release. These effects were inhibited by FK506 (10 μM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 μg/mL). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma. PMID:21964739

A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

PubMed Central

Li, Hongmei; Alavian, Kambiz N.; Lazrove, Emma; Mehta, Nabil; Jones, Adrienne; Zhang, Ping; Licznerski, Pawel; Graham, Morven; Uo, Takuma; Guo, Junhua; Rahner, Christoph; Duman, Ronald S.; Morrison, Richard S.; Jonas, Elizabeth A.

2013-01-01

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, the latter of which is dependent on mitochondrial ATP. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, however, that, Bcl-xL directly regulates endocytotic vesicle retrieval in hippocampal neurons through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytotic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity. PMID:23792689

Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

PubMed

Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

2015-09-01

Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Pegasus XL CYGNSS Departure from VAFB

NASA Image and Video Library

2016-12-02

At Vandenberg Air Force Base in California, the Orbital ATK L-1011 Stargazer, with a Pegasus XL rocket mated to the underside of the aircraft, is prepared for takeoff. On board Pegasus XL are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the /Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Departure from VAFB

NASA Image and Video Library

2016-12-02

An Orbital ATK Pegasus XL rocket is mated to the underside of the company's L-1011 Stargazer aircraft. The Stargazer is being prepared for takeoff from Vandenberg Air Force Base in California. On board Pegasus XL are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the /Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Departure from VAFB

NASA Image and Video Library

2016-12-02

The Orbital ATK L-1011 Stargazer, with a Pegasus XL rocket mated to the underside of the aircraft, takes off at sunrise from Vandenberg Air Force Base in California. On board Pegasus XL are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. The CYGNSS/Pegasus XL combination is being flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Departure from VAFB

NASA Image and Video Library

2016-12-02

The Orbital ATK L-1011 Stargazer, with a Pegasus XL rocket mated to the underside of the aircraft, has just taken off from Vandenberg Air Force Base in California. On board Pegasus XL are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. The CYGNSS/Pegasus XL combination is being flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats

PubMed Central

LIU, GUANGYI; WANG, TAO; WANG, TINGING; SONG, JINMING; ZHOU, ZHEN

2013-01-01

Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into control (n=6), sham operation (n=6) and experimental (n=48) groups. The model of rat cerebral ischemia-reperfusion injury was set up according to the method of Zea-Longa. Eight subsets of 6 rats-subset were designed according to time points (at 3, 6, 12, 24 and 48 h and at 3, 7 and 14 days). Nerve functional injury was evaluated and graded using nerve function score, balance, coordination function detection and measurement of forelimb placing. The neurons expressing caspase-3, Bax and Bcl-2 in the cortical area, CA3, CA1, stratum lucidum (Slu) and molecular layer of the dentate gyrus (MoDG) of the hippocampus were detected using immunohistochemistry or the TUNEL method. The expression of caspase-3, Bax and Bcl-2 genes was detected by the reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that, compared to the sham operation group, the score of nerve function and balance beam walking were distinctly higher (P<0.01) and the percentage of rat foreleg touching the angle or margin of the table was significantly lower in the experimental rat group (P<0.01) at 3 h following reperfusion. The expression of TUNEL-positive neurons was high in the cortical area and the CA3 region of the hippocampus (P<0.01), caspase-3 was at peak value in the cortical area and the CA1 region of the hippocampus (P<0.01), Bax was increased in the cortical area and the Slu of the hippocampus (P<0.01) and Bcl-2 was low in the cortical area and the MoDG of the hippocampus (P<0.01) in

Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats.

PubMed

Liu, Guangyi; Wang, Tao; Wang, Tinging; Song, Jinming; Zhou, Zhen

2013-11-01

Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into control (n=6), sham operation (n=6) and experimental (n=48) groups. The model of rat cerebral ischemia-reperfusion injury was set up according to the method of Zea-Longa. Eight subsets of 6 rats-subset were designed according to time points (at 3, 6, 12, 24 and 48 h and at 3, 7 and 14 days). Nerve functional injury was evaluated and graded using nerve function score, balance, coordination function detection and measurement of forelimb placing. The neurons expressing caspase-3, Bax and Bcl-2 in the cortical area, CA3, CA1, stratum lucidum (Slu) and molecular layer of the dentate gyrus (MoDG) of the hippocampus were detected using immunohistochemistry or the TUNEL method. The expression of caspase-3, Bax and Bcl-2 genes was detected by the reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that, compared to the sham operation group, the score of nerve function and balance beam walking were distinctly higher (P<0.01) and the percentage of rat foreleg touching the angle or margin of the table was significantly lower in the experimental rat group (P<0.01) at 3 h following reperfusion. The expression of TUNEL-positive neurons was high in the cortical area and the CA3 region of the hippocampus (P<0.01), caspase-3 was at peak value in the cortical area and the CA1 region of the hippocampus (P<0.01), Bax was increased in the cortical area and the Slu of the hippocampus (P<0.01) and Bcl-2 was low in the cortical area and the MoDG of the hippocampus (P<0.01) in

Inhibition of oxygen-glucose deprivation-induced apoptosis of human adipose-derived stem cells by genetic modification with antiapoptotic protein bcl-2.

PubMed

Cui, Ziwei; Shen, Liangyun; Lin, Yue; Wang, Shuqin; Zheng, Dongfeng; Tan, Qian

2014-08-01

Adipose-derived stem cells (ADSCs) have become a promising tool for a wide range of cell-based therapies. However, transplanted ADSCs do not survive well under ischemic conditions. In this study we aimed to inhibit oxygen-glucose deprivation (OGD)-induced apoptosis of human ADSCs by genetic modification with antiapoptotic protein Bcl-2. After isolation and culture, the phenotypes of human ADSCs at passage 3 were analyzed by flow cytometry. Then, genetic modification of ADSCs with Bcl-2 was carried out. Bcl-2 gene transfection was verified by Western blot analysis and multipotent differentiation properties were evaluated in Bcl-2-modified ADSCs (Bcl-2-ADSCs). Apoptosis was evaluated by a TUNEL assay under ischemic conditions induced by OGD. Apoptotic nuclei were also assessed and quantified by Hoechst staining. The cultured ADSCs expressed stem cell-associated markers CD29, CD34, CD44, and CD90, but not fibroblast marker HLA-DR or hematopoietic stem cell marker CD133. The Bcl-2 gene was transferred into ADSCs efficiently, and Bcl-2-ADSCs differentiated into adipocytes, chondrocytes, and osteoblasts. In addition, Bcl-2 overexpression reduced the percentage of apoptotic Bcl-2-ADSCs by 38 % under OGD. Our results indicate that Bcl-2 overexpression through gene transfection inhibits apoptosis of ADSCs under ischemic conditions. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

PubMed

Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

2017-08-26

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Antagonism of cytotoxic chemotherapy in neuroblastoma cell lines by 13-cis-retinoic acid is mediated by the anti-apoptotic Bcl-2 family proteins

PubMed Central

Hadjidaniel, Michael Daniel; Reynolds, Charles Patrick

2010-01-01

13-cis-retinoic acid (13-cis-RA), is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway, was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immuoblotting. Pre-treatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC99) increased from clinically achievable levels to non-achievable levels: > 5-fold (cisplatin) to > 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxiciity for neuroblastoma in vitro, mediated at least in part via the anti-apoptotic Bcl-2 family of proteins. PMID:21159604

[BCL-2 in primary central nervous system lymphomas. Immunohistochemistry and molecular biology].

PubMed

Buccoliero, A M; Castiglione, F; Caldarella, A; Rossi Degl'Innocenti, D; Taddei, A; Ammannati, F; Mennonna, P; Taddei, G L

2004-10-01

BCL-2 is a membrane protein known to be an apoptosis inhibitor. It is the product of the bcl-2 gene located on chromosome 18. Several different tumors show BCL-2 over-expression as result of a translocation or independently from it. More than 85% of follicular lymphomas and a smaller number of diffuse large cell B lymphomas contain t(14;18) (q32;q21). The aim of this study was to investigate the immunohistochemical expression of the BCL-2 protein and to ascertain, by means of traditional PCR (Polimerase Chain Reaction), its possible dependence from t(14;18) (q32;q21) in 9 primary central nervous system lymphomas. Six cases (67%) shoved immunohistochemical BCL-2 over-expression and 3 cases (33%) had t(14;18). Precisely: 2 cases (22%) had immunohistochemical BCL-2 over-expression and t(14;18) (q32;q21); 4 cases (44%) had BCL-2 over-expression without translocation; 1 case (11%) did not show diffuse BCL-2 over-expression in presence of the traslocation; the remaining 2 cases (22%) did not demonstrate BCL-2 over-expression or t(14;18) (q32;q21). In conclusion, our results indicate primary central nervous system lymphomas frequently show BCL-2 over-expression that in some case may be related to t(14;18) (q32;q21). Nevertheless, t(14;18) (q32;q21), as evaluated by traditional PCR, may not correspond to diffuse immunohistochemical BCL-2 positivity.

Ethanol Extract of Dianthus chinensis L. Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells In Vitro

PubMed Central

Nho, Kyoung Jin; Chun, Jin Mi; Kim, Ho Kyoung

2012-01-01

Dianthus chinensis L. is used to treat various diseases including cancer; however, the molecular mechanism by which the ethanol extract of Dianthus chinensis L. (EDCL) induces apoptosis is unknown. In this study, the apoptotic effects of EDCL were investigated in human HepG2 hepatocellular carcinoma cells. Treatment with EDCL significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. This induction was associated with chromatin condensation, activation of caspases, and cleavage of poly (ADP-ribose) polymerase protein. However, apoptosis induced by EDCL was attenuated by caspase inhibitor, indicating an important role for caspases in EDCL responses. Furthermore, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl. These results support a mechanism whereby EDCL induces apoptosis through the mitochondrial pathway and caspase activation in HepG2 cells. PMID:22645629

mRNA in exosomas as a liquid biopsy in non-Hodgkin Lymphoma: a multicentric study by the Spanish Lymphoma Oncology Group

PubMed Central

Rodríguez, Marta; Cantos, Blanca; Sabín, Pilar; Quero, Cristina; García-Arroyo, Francisco R.; Rueda, Antonio; Maximiano, Constanza; Rodríguez-Abreu, Delvys; Sánchez, Antonio; Silva, Javier

2017-01-01

Purpose To determine the feasibility of mRNAs (C-MYC, BCL-XL, BCL-6, NF-κβ, PTEN and AKT) in exosomes of plasma as a liquid biopsy method for monitoring and prognostic evolution in B-cell lymphomas. Patients and Methods Exosomes were isolated from 98 patients with B-cell Lymphoma and 68 healthy controls. mRNAs were analyzed by quantitative PCR. An additional 31 post-treatment samples were also studied. Results In the general and follicular lymphoma series, the presence of AKT mRNA was associated with poor response to rituximab-based treatment. Patients with first relapse or disease progression showed a lower percentage of PTEN and BCL-XL mRNA. The presence of BCL-6 mRNA was associated with a high death rate. The absence of PTEN mRNA in the general series, and presence of C-MYC mRNA in follicular lymphomas, were associated with short progression-free survival. BCL-6 and C-MYC mRNA were independent prognostic variables of overall survival. C-MYC mRNA may provide prognostic information with respect to overall survival. BCL-XL mRNA and increase of BCL-6 mRNA in post-treatment samples could serve as molecular monitoring markers. Conclusions This is the first large study to evaluate the prognostic and predictive values of pretreatment tumor-associated mRNA in exosomes. BCL-6 and C-MYC mRNA positivity in pretreatment samples were predictors of worse PFS compared to patients with mRNA negativity. C-MYC mRNA positivity was also a statistically significant predictor of inability to obtain complete response with first-line therapy. PMID:28881619

Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer.

PubMed

Li, Chunxia; Zhang, Guifeng; Zhao, Lei; Ma, Zhijun; Chen, Hongbing

2016-01-20

Nearly a century ago, Otto Warburg made the ground-breaking observation that cancer cells, unlike normal cells, prefer a seemingly inefficient mechanism of glucose metabolism: aerobic glycolysis, a phenomenon now referred to as the Warburg effect. The finding that rapidly proliferating cancer cells favors incomplete metabolism of glucose, producing large amounts of lactate as opposed to synthesizing ATP to sustain cell growth, has confounded scientists for years. Further investigation into the metabolic phenotype of cancer has expanded our understanding of this puzzling conundrum, and has opened new avenues for the development of anti-cancer therapies. Enhanced glycolytic flux is now known to allow for increased synthesis of intermediates for sustaining anabolic pathways critical for cancer cell growth. Alongside the increase in glycolysis, cancer cells transform their mitochondria into synthesis machines supported by augmented glutaminolysis, supplying lipid production, amino acid synthesis, and the pentose phosphate pathways. Inhibition of several of the key enzymes involved in these pathways has been demonstrated to effectively obstruct cancer cell growth and multiplication, sensitizing them to apoptosis. The modulation of various regulatory proteins involved in metabolic processes is central to cancerous reprogramming of metabolism. The finding that members of one of the major protein families involved in cell death regulation also aberrantly regulated in cancers, the Bcl-2 family of proteins, are also critical mediators of metabolic pathways, provides strong evidence for the importance of the metabolic shift to cancer cell survival. Targeting the anti-apoptotic members of the Bcl-2 family of proteins is proving to be a successful way to selectively target cancer cells and induce apoptosis. Further understanding of how cancer cells modify metabolic regulation to increase channeling of substrates into biosynthesis will allow for the discovery of novel drug

Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting▿ †

PubMed Central

Leski, Tomasz A.; Caswell, Clayton C.; Pawlowski, Marcin; Klinke, David J.; Bujnicki, Janusz M.; Hart, Sean J.; Lukomski, Slawomir

2009-01-01

The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms. PMID:19767469

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

PubMed

Josefsson, Emma C; Burnett, Deborah L; Lebois, Marion; Debrincat, Marlyse A; White, Michael J; Henley, Katya J; Lane, Rachael M; Moujalled, Diane; Preston, Simon P; O'Reilly, Lorraine A; Pellegrini, Marc; Metcalf, Donald; Strasser, Andreas; Kile, Benjamin T

2014-03-17

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

Bcl-2-like Protein 11 (BIM) Expression Is Associated with Favorable Prognosis for Patients with Cervical Cancer.

PubMed

Kim, Bo Wook; Cho, Hanbyoul; Ylaya, Kris; Kitano, Haruhisa; Chung, Joon-Yong; Hewitt, Stephen M; Kim, Jae-Hoon

2017-09-01

Bcl-2-like protein 11 (BIM) is a pro-apoptotic member of the Bcl-2 protein family. BIM elicits cell death by binding to pro-survival Bcl-2 proteins. Even though the association of BIM expression with cell death has been investigated, its clinical survival significance in cervical cancer has not. In the current study, the prognostic significance of BIM in cervical cancer was investigated. The study included normal cervical tissues (n=254), cervical intraepithelial neoplasia (CIN) tissues (n=275), and invasive cervical cancer (n=164). In order to identify BIM expression, immunohistochemistry (IHC) was performed, and IHC scoring by quantitative digital image analysis was determined. Then, the association of BIM with prognostic factors was investigated. BIM expression was higher in cervical cancer than normal cervical tissues (p<0.001). Well and moderate differentiation indicated higher BIM expression than did poor differentiation (p=0.001). Also, BIM expression was high in radiation-sensitive cervical cancer relative to radiation-resistant cancer (p=0.049). High BIM expression showed better 5-year disease-free survival (DFS) and overall survival (OS) rates (p=0.049 and π=0.030, respectively) than did low expression. In a multivariate analysis, BIM was shown to be an independent risk factor for DFS and OS in cervical cancer, with hazard ratios of 0.22 (p=0.006) and 0.46 (p=0.046), respectively. BIM is associated with favorable prognostic markers for prediction of DFS and OS in cervical cancer. High BIM expression is a potential prognostic marker as well as a chemotherapeutic target for cervical cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

High expression of anti-apoptotic protein Bcl-2 is a good prognostic factor in colorectal cancer: Result of a meta-analysis.

PubMed

Huang, Qi; Li, Shu; Cheng, Pu; Deng, Mei; He, Xin; Wang, Zhen; Yang, Cheng-Hui; Zhao, Xiao-Ying; Huang, Jian

2017-07-21

To systematically evaluate the prognostic-predictive capability of Bcl-2 in colorectal cancer (CRC). A systematic literature search was conducted using PubMed, Web of Science and EMBASE databases. Any eligible study must meet the following criteria: (1) bcl-2 expression was evaluated in human CRC tissues by immunohistochemistry; (2) assessment of the relationships between bcl-2 expression and overall survival (OS), disease free survival (DFS), recurrent free survival (RFS) or clinic-pathological characteristics of CRC was included; (3) sufficient information was provided to estimate the hazard ratio (HR) or odds ratio and their 95% confidence intervals (CIs); and (4) the study was published in English. The impact of Bcl-2 expression on survival of CRC patients were evaluated through this meta-analysis. A total of 40 eligible articles involving 7658 patients were enrolled in our final analysis. We drew the conclusion that Bcl-2 high expression was significantly correlated with favorable OS (pooled HR = 0.69, 95%CI: 0.55-0.87, P = 0.002) and better DFS/RFS (pooled HR = 0.65, 95%CI: 0.50-0.85, P = 0.001). Additionally, the subgroup analysis suggested that Bcl-2 overexpression was significantly associated with prognosis (OS) especially in patients came from Europe and America but not Asian and patients who did not receive any adjuvant therapy before surgery. Finally, our present results indicated that expression of bcl-2 protein was associated with high differentiation grade and A/B Ducks' stage. Bcl-2 high expression was significantly correlated with favorable OS and better DFS/RFS. Hence, we propose that Bcl-2 may be a valuable prognostic-predictive marker in CRC.

High expression of anti-apoptotic protein Bcl-2 is a good prognostic factor in colorectal cancer: Result of a meta-analysis

PubMed Central

Huang, Qi; Li, Shu; Cheng, Pu; Deng, Mei; He, Xin; Wang, Zhen; Yang, Cheng-Hui; Zhao, Xiao-Ying; Huang, Jian

2017-01-01

AIM To systematically evaluate the prognostic-predictive capability of Bcl-2 in colorectal cancer (CRC). METHODS A systematic literature search was conducted using PubMed, Web of Science and EMBASE databases. Any eligible study must meet the following criteria: (1) bcl-2 expression was evaluated in human CRC tissues by immunohistochemistry; (2) assessment of the relationships between bcl-2 expression and overall survival (OS), disease free survival (DFS), recurrent free survival (RFS) or clinic-pathological characteristics of CRC was included; (3) sufficient information was provided to estimate the hazard ratio (HR) or odds ratio and their 95% confidence intervals (CIs); and (4) the study was published in English. The impact of Bcl-2 expression on survival of CRC patients were evaluated through this meta-analysis. RESULTS A total of 40 eligible articles involving 7658 patients were enrolled in our final analysis. We drew the conclusion that Bcl-2 high expression was significantly correlated with favorable OS (pooled HR = 0.69, 95%CI: 0.55-0.87, P = 0.002) and better DFS/RFS (pooled HR = 0.65, 95%CI: 0.50-0.85, P = 0.001). Additionally, the subgroup analysis suggested that Bcl-2 overexpression was significantly associated with prognosis (OS) especially in patients came from Europe and America but not Asian and patients who did not receive any adjuvant therapy before surgery. Finally, our present results indicated that expression of bcl-2 protein was associated with high differentiation grade and A/B Ducks’ stage. CONCLUSION Bcl-2 high expression was significantly correlated with favorable OS and better DFS/RFS. Hence, we propose that Bcl-2 may be a valuable prognostic-predictive marker in CRC. PMID:28785155

Bcl-2 family of proteins as drug targets for cancer chemotherapy: the long way of BH3 mimetics from bench to bedside.

PubMed

Vela, Laura; Marzo, Isabel

2015-08-01

Bcl-2 proteins are key determinants in the life-death balance. In recent years, proteins in this family have been identified as drug targets in the design of new anti-tumor therapies. Advances in the knowledge of the mechanism of action of anti-apoptotic and pro-apoptotic members of the Bcl-2 family have enabled the development of the so-called 'BH3 mimetics'. These compounds act by inhibiting anti-apoptotic proteins of the family, imitating the function of the BH3-only subset of pro-apoptotic members. Combinations of BH3-mimetics with anti-tumor drugs are being evaluated in both preclinical models and clinical trials. Recent advances in these approaches will be reviewed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Progress in BCL2 inhibition for patients with chronic lymphocytic leukemia.

PubMed

Tam, Constantine S; Seymour, John F; Roberts, Andrew W

2016-04-01

The prosurvival protein BCL2 is uniformly expressed in chronic lymphocytic leukemia (CLL), and enables leukemia cell survival in the face of cytotoxic treatment and increasing genomic, metabolic, and oxidative stresses. The therapeutic potential of BCL2 inhibition was first observed in the clinic following BCL2 antisense therapy. Subsequently, a number of small molecule inhibitors were developed to mimic the function of the pro-apoptotic BH3-only proteins (BH3-mimetics). These molecules are now in late-phase clinical trials and demonstrate potent activity, including the occurrence of acute tumor lysis syndrome in subjects with multiply relapsed, chemorefractory CLL. In this review, we discuss the history and summarize current knowledge regarding BCL2 inhibition as therapy of CLL. Copyright © 2016 Elsevier Inc. All rights reserved.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

[Survival of patients with primary central nervous system diffuse large B-cell lymphoma: impact of gene aberrations and protein overexpression of bcl-2 and C-MYC, and selection of chemotherapy regimens].

PubMed

Yin, W J; Zhu, X; Yang, H Y; Sun, W Y; Wu, M J

2018-01-08

Objective: To investigate the impact of clinicopathological features, gene rearrangements and protein expression of bcl-6, bcl-2, C-MYC and chemotherapy regime on the prognosis of patients with primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL). Methods: Thirty-three cases of PCNS-DLBCL diagnosed from January 2006 to December 2016 at Zhejiang Cancer Hospital were collected. The expression of CD10, bcl-6, bcl-2, MUM1 and MYC were detected by immunohistochemical staining (IHC). The presence of EB virus was detected by in situ hybridization(EBER). Copy number variation (ICN) and translocation status of bcl-6, bcl-2 and C-MYC genes were detected by fluorescence in situ hybridization (FISH). The relationship between the above indexes and the prognosis was analyzed by univariate, bivariate survival analysis and multiple Cox hazard regression analysis. Results: The study included 33 patients of PCNS-DLBCL, without evidence of primary or secondary immunodeficient disease. Male to female ratio was 1.36∶1.00, and the average age was 56 years. Twenty cases had single lesion while 13 had multiple lesions. Deep brain involvement was seen in 12 cases. All patients underwent partial or total tumor resection. Five patients received whole brain post-surgery radiotherapy, nine patients received high-dose methotrexate (HD-MTX) based chemotherapy, and 12 patients received whole-brain radiotherapy combined with HD-MTX based chemotherapy. Severn patients received no further treatment and rituximab was used in 8 patients. According to the Hans model, 27 cases were classified as non-GCB subtypes (81.8%). Bcl-2 was positive in 25 cases (75.8%, 25/33) and highly expressed in 8 (24.2%). MYC was positive in 12 cases (36.4%) and double expression of bcl-2 and MYC was seen in 6 cases. EBER positive rate was 10.0%(3/30), all of which had multiple lesions. Two bcl-6 gene translocations and 3 amplifications were found in 28 patients. Two translocations, 3 ICN or with both

The role of the acidity of N-heteroaryl sulfonamides as inhibitors of bcl-2 family protein-protein interactions.

PubMed

Touré, B Barry; Miller-Moslin, Karen; Yusuff, Naeem; Perez, Lawrence; Doré, Michael; Joud, Carol; Michael, Walter; DiPietro, Lucian; van der Plas, Simon; McEwan, Michael; Lenoir, Francois; Hoe, Madelene; Karki, Rajesh; Springer, Clayton; Sullivan, John; Levine, Kymberly; Fiorilla, Catherine; Xie, Xiaoling; Kulathila, Raviraj; Herlihy, Kara; Porter, Dale; Visser, Michael

2013-02-14

Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263.

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

PubMed

Meller, R; Schindler, C K; Chu, X P; Xiong, Z G; Cameron, J A; Simon, R P; Henshall, D C

2003-05-01

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

The IκB family member Bcl-3 coordinates the pulmonary defense against Klebsiella pneumoniae infection.

PubMed

Pène, Frédéric; Paun, Andrea; Sønder, Søren Ulrik; Rikhi, Nimisha; Wang, Hongshan; Claudio, Estefania; Siebenlist, Ulrich

2011-02-15

Bcl-3 is an atypical member of the IκB family that has the potential to positively or negatively modulate nuclear NF-κB activity in a context-dependent manner. Bcl-3's biologic impact is complex and includes roles in tumorigenesis and diverse immune responses, including innate immunity. Bcl-3 may mediate LPS tolerance, suppressing cytokine production, but it also seems to contribute to defense against select systemic bacterial challenges. However, the potential role of Bcl-3 in organ-specific host defense against bacteria has not been addressed. In this study, we investigated the relevance of Bcl-3 in a lung challenge with the Gram-negative pathogen Klebsiella pneumoniae. In contrast to wild-type mice, Bcl-3-deficient mice exhibited significantly increased susceptibility toward K. pneumoniae pneumonia. The mutant mice showed increased lung damage marked by neutrophilic alveolar consolidation, and they failed to clear bacteria in lungs, which correlated with increased bacteremic dissemination. Loss of Bcl-3 incurred a dramatic cytokine imbalance in the lungs, which was characterized by higher levels of IL-10 and a near total absence of IFN-γ. Moreover, Bcl-3-deficient mice displayed increased lung production of the neutrophil-attracting chemokines CXCL-1 and CXCL-2. Alveolar macrophages and neutrophils are important to antibacterial lung defense. In vitro stimulation of Bcl-3-deficient alveolar macrophages with LPS or heat-killed K. pneumoniae recapitulated the increase in IL-10 production, and Bcl-3-deficient neutrophils were impaired in intracellular bacterial killing. These findings suggest that Bcl-3 is critically involved in lung defense against Gram-negative bacteria, modulating functions of several cells to facilitate efficient clearance of bacteria.

Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

PubMed

Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

2017-06-01

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Pharmacological and protein profiling suggest venetoclax (ABT-199) as optimal partner with ibrutinib in chronic lymphocytic leukemia

PubMed Central

Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A.; Wierda, William G.; Keating, Michael J.; Balakrishnan, Kumudha; Gandhi, Varsha

2015-01-01

Purpose Bruton’s tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Experimental design Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199) and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. Results The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted highly cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family anti-apoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Conclusions Our biological and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. PMID:25829398

Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

PubMed

Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A; Wierda, William G; Keating, Michael J; Balakrishnan, Kumudha; Gandhi, Varsha

2015-08-15

Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. ©2015 American Association for Cancer Research.

Pegasus XL CYGNSS Fairing Installation

NASA Image and Video Library

2016-11-11

At Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is seen during payload fairing installation in Building 1555. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fairing Installation

NASA Image and Video Library

2016-11-11

In Building 1555 at Vandenberg Air Force Base in California, the payload fairing is being installed on an Orbital ATK Pegasus XL rocket. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Reactive ion etching of GaN using BCl 3, BCl 3/Ar and BCl 3/ N 2 gas plasmas

NASA Astrophysics Data System (ADS)

Basak, D.; Nakanishi, T.; Sakai, S.

2000-04-01

Reactive ion etching (RIE) of GaN has been performed using BCl 3 and additives, Ar and N 2, to BCl 3 plasma. The etch rate, surface roughness and the etch profile have been investigated. The etch rate of GaN is found to be 104 nm/min at rf power of 200 W, pressure of 2 Pa, with 9.5 sccm flow rate of BCl 3. The addition of 5 sccm of Ar to 9.5 sccm of BCl 3 reduces the etch rate of GaN while the addition of N 2 does not influence the etch rate significantly. The RIE of GaN layer with BCl 3/Ar and BCl 3/N 2 results in a smoother surface compared to surfaces etched with BCl 3 only. The etched side-wall in BCl 3 plasma makes an angle of 60° with the normal surface, and the angle of inclination is more in cases of BCl 3/Ar and BCl 3/N 2 plasmas. The RIE induced damage to the surface is measured qualitatively by PL measurements. It is observed that the damage to the etched surfaces is similar for all the plasmas.

Paraffin immunoreactivity of CD10, CDw75, and Bcl-6 in follicle center cell lymphoma.

PubMed

Dunphy, C H; Polski, J M; Lance Evans, H; Gardner, L J

2001-05-01

Follicle center cell lymphoma(FCCL) has the following immunophenotype(IP): sIg+, Pan B+, CD10+/-, CD5-, CD23-/+, CD43-, CD11c-, CD25-. In addition, reactivities of a malignant lymphoma with CDw75(LN-1) and bcl-6 are considered indicators of FCCL. Bcl-6 expression is common in Grade 1 FCCL (100%) and rare in other indolent B-cell lymphomas(BCL). In contrast, bcl-2 expression is common in FCCL (80%) and in other BCL subtypes. Since no previous study has correlated paraffin immunoreactivity(PIR) of CD10, CDw75, and bcl-6 in FCCL (Grades 1-3), this is this study's purpose. Twenty-nine FCCL's were identified and reviewed (6, Grade 1; 10, Grade 2; 13, Grade 3) from the Division of Hematopathology, St. Louis University. The diagnoses were based on morphology and immunohistochemistry(IH)(21 cases) +/- the flow cytometric IP(14 cases). The paraffin blocks were stained for CD10 (Novacastra, Vector Laboratories, Burlingame, CA), CDw75 and bcl-6 (DAKO Corporation, Carpinteria, CA). Results showed that, CD10 by paraffin IH(PIH) was positive in 23 [18(strong); 3(moderate); 2(weak)] and negative in 6(3, Grade 2; 3, Grade 3). All CD10-cases were CDw75+; 4, bcl-6+. The two CD10-, bcl-6-cases were Grade 2. CDw75 was positive in 28 cases [16(strong); 11(moderate); 1(weak)] and negative in 1 (Grade 3; CD10+, bcl-2+, bcl-6+). Bcl-6 was positive in 26 [16(strong); 6(moderate); 4(weak)] and negative in 3(Grade 2's). Thus, the sensitivity of CD10, CDw75, and bcl-6 by PIH for FCCL was 79%, 97%, and 90%, respectively. Of the three stains evaluated by PIH in FCCL, CDw75 was the most sensitive, closely followed by bcl-6. CD10 was least sensitive-79%. By combining these 3 stains, the sensitivity was 100%; thus, a combined approach is recommended.

MCL-1–dependent leukemia cells are more sensitive to chemotherapy than BCL-2–dependent counterparts

PubMed Central

Brunelle, Joslyn K.; Ryan, Jeremy; Yecies, Derek; Opferman, Joseph T.

2009-01-01

Myeloid cell leukemia sequence 1 (MCL-1) and B cell leukemia/lymphoma 2 (BCL-2) are anti-apoptotic proteins in the BCL-2 protein family often expressed in cancer. To compare the function of MCL-1 and BCL-2 in maintaining cancer survival, we constructed complementary mouse leukemia models based on Eμ-Myc expression in which either BCL-2 or MCL-1 are required for leukemia maintenance. We show that the principal anti-apoptotic mechanism of both BCL-2 and MCL-1 in these leukemias is to sequester pro-death BH3-only proteins rather than BAX and BAK. We find that the MCL-1–dependent leukemias are more sensitive to a wide range of chemotherapeutic agents acting by disparate mechanisms. In common across these varied treatments is that MCL-1 protein levels rapidly decrease in a proteosome-dependent fashion, whereas those of BCL-2 are stable. We demonstrate for the first time that two anti-apoptotic proteins can enable tumorigenesis equally well, but nonetheless differ in their influence on chemosensitivity. PMID:19948485

Pegasus XL CYGNSS Mate to L-1011

NASA Image and Video Library

2016-11-28

At Vandenberg Air Force Base in California, the Orbital ATK L-1011 Stargazer awaits a Pegasus XL rocket to be mated to the aircraft. On board Pegasus XL are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the /Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

PubMed

de Graaf, Aniek O; van den Heuvel, Lambert P; Dijkman, Henry B P M; de Abreu, Ronney A; Birkenkamp, Kim U; de Witte, Theo; van der Reijden, Bert A; Smeitink, Jan A M; Jansen, Joop H

2004-10-01

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer.

PubMed

Goodwin, C M; Rossanese, O W; Olejniczak, E T; Fesik, S W

2015-12-01

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency

Venetoclax responses of pediatric ALL xenografts reveal sensitivity of MLL-rearranged leukemia.

PubMed

Khaw, Seong Lin; Suryani, Santi; Evans, Kathryn; Richmond, Jennifer; Robbins, Alissa; Kurmasheva, Raushan T; Billups, Catherine A; Erickson, Stephen W; Guo, Yuelong; Houghton, Peter J; Smith, Malcolm A; Carol, Hernan; Roberts, Andrew W; Huang, David C S; Lock, Richard B

2016-09-08

The clinical success of the BCL-2-selective BH3-mimetic venetoclax in patients with poor prognosis chronic lymphocytic leukemia (CLL) highlights the potential of targeting the BCL-2-regulated apoptotic pathway in previously untreatable lymphoid malignancies. By selectively inhibiting BCL-2, venetoclax circumvents the dose-limiting, BCL-XL-mediated thrombocytopenia of its less selective predecessor navitoclax, while enhancing efficacy in CLL. We have previously reported the potent sensitivity of many high-risk childhood acute lymphoblastic leukemia (ALL) xenografts to navitoclax. Given the superior tolerability of venetoclax, here we have investigated its efficacy in childhood ALL. We demonstrate that in contrast to the clear dependence of CLL on BCL-2 alone, effective antileukemic activity in the majority of ALL xenografts requires concurrent inhibition of both BCL-2 and BCL-XL We identify BCL-XL expression as a key predictor of poor response to venetoclax and demonstrate that concurrent inhibition of both BCL-2 and BCL-XL results in synergistic killing in the majority of ALL xenografts. A notable exception is mixed lineage leukemia-rearranged infant ALL, where venetoclax largely recapitulates the activity of navitoclax, identifying this subgroup of patients as potential candidates for clinical trials of venetoclax in childhood ALL. Conversely, our findings provide a clear basis for progressing navitoclax into trials ahead of venetoclax in other subgroups.

Venetoclax responses of pediatric ALL xenografts reveal sensitivity of MLL-rearranged leukemia

PubMed Central

Khaw, Seong Lin; Suryani, Santi; Evans, Kathryn; Richmond, Jennifer; Robbins, Alissa; Kurmasheva, Raushan T.; Billups, Catherine A.; Erickson, Stephen W.; Guo, Yuelong; Houghton, Peter J.; Smith, Malcolm A.; Carol, Hernan; Roberts, Andrew W.; Huang, David C. S.

2016-01-01

The clinical success of the BCL-2-selective BH3-mimetic venetoclax in patients with poor prognosis chronic lymphocytic leukemia (CLL) highlights the potential of targeting the BCL-2-regulated apoptotic pathway in previously untreatable lymphoid malignancies. By selectively inhibiting BCL-2, venetoclax circumvents the dose-limiting, BCL-XL-mediated thrombocytopenia of its less selective predecessor navitoclax, while enhancing efficacy in CLL. We have previously reported the potent sensitivity of many high-risk childhood acute lymphoblastic leukemia (ALL) xenografts to navitoclax. Given the superior tolerability of venetoclax, here we have investigated its efficacy in childhood ALL. We demonstrate that in contrast to the clear dependence of CLL on BCL-2 alone, effective antileukemic activity in the majority of ALL xenografts requires concurrent inhibition of both BCL-2 and BCL-XL. We identify BCL-XL expression as a key predictor of poor response to venetoclax and demonstrate that concurrent inhibition of both BCL-2 and BCL-XL results in synergistic killing in the majority of ALL xenografts. A notable exception is mixed lineage leukemia–rearranged infant ALL, where venetoclax largely recapitulates the activity of navitoclax, identifying this subgroup of patients as potential candidates for clinical trials of venetoclax in childhood ALL. Conversely, our findings provide a clear basis for progressing navitoclax into trials ahead of venetoclax in other subgroups. PMID:27343252

Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis

PubMed Central

Ryder, Christopher; McColl, Karen; Zhong, Fei; Distelhorst, Clark W.

2012-01-01

Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway. PMID:22685289

Nuclear NF-kappaB p65 phosphorylation at serine 276 by protein kinase A contributes to the malignant phenotype of head and neck cancer.

PubMed

Arun, Pattatheyil; Brown, Matthew S; Ehsanian, Reza; Chen, Zhong; Van Waes, Carter

2009-10-01

Aberrant nuclear activation and phosphorylation of the canonical NF-kappaB subunit RELA/p65 at Serine-536 by inhibitor kappaB kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-kappaB activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by protein kinase A (PKA) in the malignant phenotype and gene transactivation, and studied p65-Ser276 as a potential target for therapy. Phospho and total p65 protein expression and localization were determined in HNSCC tissue array and in cell lines. The effects of the PKA inhibitor H-89 on NF-kappaB activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity. NF-kappaB p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, tumor necrosis factor-alpha (TNF-alpha) significantly increased, whereas H-89 inhibited constitutive and TNF-alpha-induced nuclear p65 (Ser276) phosphorylation, and significantly suppressed NF-kappaB and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-kappaB, IL-8, and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death, and blocked the cell cycle in G(1)-S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-kappaB-related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21(CIP1/WAF1), while suppressing proliferative marker Ki67. NF-kappaB p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC.

From molecular PDT damage to cellular PDT responses: attempts at bridging the gap on the role of Bcl-2

NASA Astrophysics Data System (ADS)

Usuda, Jitsuo; Xue, Liang-yan; Chiu, Song-mao; Azizuddin, Kashif; Morris, Rachel L.; Mulvihill, John; Oleinick, Nancy L.

2003-06-01

Expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL is greatly elevated in many advanced cancers, especially those resistant to standard therapies, such as radiation or chemotherapy. It has been suggested that those two proteins would be attractive targets for the development of new cancer treatments. Photodynamic therapy (PDT) with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, specifically attack the anti-apoptotic protein Bcl-2, generating a variety of oxidized, complexed, and cleaved photoproducts. The closely related protein Bcl-xL is also a target of Pc 4-PDT. In a recent study employing transient transfection of an expression vector encoding deletion mutants of Bcl-2, we identified the membrane anchorage regions of the protein that are required to form the photosensitive target. In spite of the demonstrated photodamage to Bcl-2 (and Bcl-xL), how the photodamage translates into changes in the sensitivity of cells to PDT-induced apoptosis or other modes of cell death is not clear, and it also remains unclear how elevated amounts of anti-apoptotic proteins in tumors might make them more or less responsive to PDT. In the present study, we have studied the PDT response of MCF7 human breast cancer cells overexpressing wild-type Bcl-2 or certain deletion mutants either in a transient or stable mode. We show that cells expressing modestly elevated amounts (<10-fold increase) of Bcl-2 and in which the pro-apoptotic protein Bax is not upregulated do not differ from the parental cells with respect to PDT-induced cell killing. In contrast, cells expressing higher amounts (>50-fold increase) of Bcl-2 or certain mutants are made significantly more resistant to the induction of apoptosis and the loss of clonogenicity upon exposure to Pc 4-PDT. In the presence of high levels of Bcl-2, extensive photodamage requires higher PDT doses. We conclude that Pc 4-PDT targets Bcl-2 and Bcl-xL, eliminating one mechanism that

Jinhong Tablet Reduces Damage of Intestinal Mucosal Barrier in Rats with Acute Biliary Infection via Bcl-2/Bax mRNA and Protein Regulation

PubMed Central

Wang, YongQi; Xie, Jinkun; Zhang, Xuelin; Gu, Honggang

2017-01-01

Objective To explore the effects and mechanism of Jinhong Tablet on intestinal mucosal barrier function and SIRS in rats with acute biliary infection. Methods 36 SD male rats were divided into three groups: sham operation (control), acute biliary infection (ABI) model, and Jinhong Tablet (Jinhong) group. Jinhong group were force-fed with Jinhong Tablet, while the other two groups received oral saline. At days 3 and 5, morphological changes of intestinal mucosa were assessed. Serum diamine oxidase (DAO), D-lactate, and endotoxin levels were measured. And the genes bcl-2 and bax in intestinal tissues were tested by real-time PCR and Western blotting. Results Intestinal damage was significantly less severe in Jinhong group compared with ABI group, as indicated by Chiu's scoring, TUNEL analysis, and serum DAO, D-lactic acid, and endotoxin levels. Additionally, the expression of bax mRNA and protein was decreased and the ratio of bcl-2/bax mRNA and protein was increased compared with ABI group. Conclusion Jinhong Tablet had a positive intervention on acute biliary infection through improving inflammation and intestinal mucosal barrier, inhibiting excessive apoptosis of intestinal epithelial cells via bax and bcl-2 gene, and protein regulation. PMID:29234407

FADD and the NF-κB family member Bcl-3 regulate complementary pathways to control T-cell survival and proliferation

PubMed Central

Rangelova, Svetla; Kirschnek, Susanne; Strasser, Andreas; Häcker, Georg

2008-01-01

Fas-associated protein with death domain/mediator of receptor induced toxicity (FADD/MORT1) was first described as a transducer of death receptor signalling but was later recognized also to be important for proliferation of T cells. B-cell lymphoma 3 (Bcl-3) is a relatively little understood member of the nuclear factor (NF)-κB family of transcription factors. We recently found that Bcl-3 is up-regulated in T cells from mice where FADD function is blocked by a dominant negative transgene (FADD-DN). To understand the importance of this, we generated FADD-DN/bcl-3−/− mice. Here, we report that T cells from these mice show massive cell death and severely reduced proliferation in response to T-cell receptor (TCR) stimulation in vitro. Transgenic co-expression of Bcl-2 (FADD-DN/bcl-3−/−/vav-bcl-2 mice) rescued the survival but not the proliferation of T cells. FADD-DN/bcl-3−/− mice had normal thymocyte numbers but reduced numbers of peripheral T cells despite an increase in cycling T cells in vivo. However, activation of the classical NF-κB and extracellular regulated kinase (ERK) pathways and expression of interleukin (IL)-2 mRNA upon stimulation were normal in T cells from FADD-DN/bcl-3−/− mice. These data suggest that FADD and Bcl-3 regulate separate pathways that both contribute to survival and proliferation in mouse T cells. PMID:18557791

Phosphorylation status modulates Bcl-2 function during glucocorticoid-induced apoptosis in T lymphocytes.

PubMed

Huang, Se-Te J; Cidlowski, John A

2002-06-01

Glucocorticoids are known to induce apoptosis in lymphoid cells, and Bcl-2 overexpression can block the apoptosis-inducing action of glucocorticoids. Since phosphorylation of Bcl-2 is implicated in regulating Bcl-2 function, we considered the role of Bcl-2 phosphorylation in protecting lymphoid cells from glucocorticoid-induced cell death. Five stably transfected cell lines of WEHI 7.1 cells expressing either wild-type Bcl-2 or alanine mutants of Bcl-2 at amino acids threonine 56, serine 70, threonine 74, or serine 87 were created. Expression of the mutant Bcl-2 proteins was documented by flow cytometry and Western blot analysis. Mutation of Bcl-2 on T56 and S87 eliminated the ability of Bcl-2 to inhibit glucocorticoid-induced cell shrinkage, mitochondrial depolarization, DNA fragmentation, and cell death. Mutation of T74 only partially impaired the ability of Bcl-2 to block glucocorticoid-induced apoptosis whereas mutation of S70 in Bcl-2 did not alter its ability to block glucocorticoid-induced apoptosis.

Bcl11b-A Critical Neurodevelopmental Transcription Factor-Roles in Health and Disease.

PubMed

Lennon, Matthew J; Jones, Simon P; Lovelace, Michael D; Guillemin, Gilles J; Brew, Bruce J

2017-01-01

B cell leukemia 11b (Bcl11b) is a zinc finger protein transcription factor with a multiplicity of functions. It works as both a genetic suppressor and activator, acting directly, attaching to promoter regions, as well as indirectly, attaching to promoter-bound transcription factors. Bcl11b is a fundamental transcription factor in fetal development, with important roles for the differentiation and development of various neuronal subtypes in the central nervous system (CNS). It has been used as a specific marker of layer V subcerebral projection neurons as well as striatal interneurons. Bcl11b also has critical developmental functions in the immune, integumentary and cardiac systems, to the extent that Bcl11b knockout mice are incompatible with extra-uterine life. Bcl11b has been implicated in a number of disease states including Huntington's disease, Alzheimer's disease, HIV and T-Cell malignancy, amongst others. Bcl11b is a fascinating protein whose critical roles in the CNS and other parts of the body are yet to be fully explicated. This review summarizes the current literature on Bcl11b and its functions in development, health, and disease as well as future directions for research.

BCL-2 as therapeutic target for hematological malignancies.

PubMed

Perini, Guilherme Fleury; Ribeiro, Glaciano Nogueira; Pinto Neto, Jorge Vaz; Campos, Laura Tojeiro; Hamerschlak, Nelson

2018-05-11

Disruption of the physiologic balance between cell proliferation and cell death is an important step of cancer development. Increased resistance to apoptosis is a key oncogenic mechanism in several hematological malignancies and, in many cases, especially in lymphoid neoplasias, has been attributed to the upregulation of BCL-2. The BCL-2 protein is the founding member of the BCL-2 family of apoptosis regulators and was the first apoptosis modulator to be associated with cancer. The recognition of the important role played by BCL-2 for cancer development and resistance to treatment made it a relevant target for therapy for many diseases, including solid tumors and hematological neoplasias. Among the different strategies that have been developed to inhibit BCL-2, BH3-mimetics have emerged as a novel class of compounds with favorable results in different clinical settings, including chronic lymphocytic leukemia (CLL). In April 2016, the first inhibitor of BCL-2, venetoclax, was approved by the US Food and Drug Administration for the treatment of patients with CLL who have 17p deletion and had received at least one prior therapy. This review focuses on the relevance of BCL-2 for apoptosis modulation at the mitochondrial level, its potential as therapeutic target for hematological malignancies, and the results obtained with selective inhibitors belonging to the BH3-mimetics, especially venetoclax used in monotherapy or in combination with other agents.

Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

PubMed

Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

2014-03-01

Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Bcl-2 antisense therapy in B-cell malignant proliferative disorders.

PubMed

Chanan-Khan, Asher; Czuczman, Myron S

2004-08-01

Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.

Discovery and molecular characterization of a Bcl-2-regulated cell death pathway in schistosomes.

PubMed

Lee, Erinna F; Clarke, Oliver B; Evangelista, Marco; Feng, Zhiping; Speed, Terence P; Tchoubrieva, Elissaveta B; Strasser, Andreas; Kalinna, Bernd H; Colman, Peter M; Fairlie, W Douglas

2011-04-26

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

Molecular basis for the interplay of apoptosis and proliferation mediated by Bcl-xL:Bim interactions in pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abrol, Ravinder, E-mail: abrol@wag.caltech.edu; Edderkaoui, Mouad; Goddard, William A.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Direct role of Bcl-2 protein interactions in cell proliferation is not clear. Black-Right-Pointing-Pointer Designed Bcl-xL mutants show opposite effects on apoptosis and proliferation. Black-Right-Pointing-Pointer Disrupting Bcl-xL:Bim interaction increased apoptosis in pancreatic cancer. Black-Right-Pointing-Pointer Disrupting Bcl-xL:Bim interaction decreased proliferation in pancreatic cancer. Black-Right-Pointing-Pointer Bcl-xL:Bim interaction can control both apoptosis and proliferation. -- Abstract: A major mechanism through which cancer cells avoid apoptosis is by promoting the association of anti-apoptotic members of the pro-survival Bcl-2 protein family (like Bcl-2 and Bcl-xL) with BH{sub 3} domain-only proteins (like Bim and Bid). Apoptosis and cell proliferation have been shown to be linkedmore » for many cancers but the molecular basis for this link is far from understood. We have identified the Bcl-xL:Bim protein-protein interface as a direct regulator of proliferation and apoptosis in pancreatic cancer cells. We were able to predict and subsequently verify experimentally the effect of various Bcl-xL single-point mutants (at the position A142) on binding to Bim by structural analysis and computational modeling of the inter-residue interactions at the Bcl-xL:Bim protein-protein interface. The mutants A142N, A142Q, and A142Y decreased binding of Bim to Bcl-xL and A142S increased this binding. The Bcl-xL mutants, with decreased affinity for Bim, caused an increase in apoptosis and a corresponding decrease in cell proliferation. However, we could prevent these effects by introducing a small interfering RNA (siRNA) targeted at Bim. These results show a novel role played by the Bcl-xL:Bim interaction in regulating proliferation of pancreatic cancer cells at the expense of apoptosis. This study presents a physiologically relevant model of the Bcl-xL:Bim interface that can be used for rational therapeutic design for

[Knock-down of BCL11A expression in breast cancer cells promotes MDA-MB-231 cell apoptosis].

PubMed

Li, Hongli; Gui, Chen; Yan, Lijun

2016-11-01

Objective To detect the expression and pathological significance of B-cell CLL/lymphoma 11A (BCL11A) in breast cancer and investigate the effect of its silencing on the apoptosis of human MDA-MB-231 breast cancer cells. MethodsImmunohistochemistry was used to detect the expression of BCL11A in 62 cases of human breast cancer tissues and 8 cases of normal tissues. We synthesized siRNA targeting BCL11A, and then siRNA was transfected into MDA-MB-231 cells. Forty-eight hours later, the suppression effect of siRNA on BCL11A was determined by quantitative real-time PCR and Western blotting. The apoptosis of MDA-MB-231 cells was detected by flow cytometry. Results The BCL11A protein was mainly expressed in cytoplasm. The expression level of BCL11A in breast cancer tissues was higher than that in paracancerous tissues. The expression had correlations with tumor grade, tumor stage, while it had no correlations with the patients' age and tumor size. BCL11A-siRNA significantly suppressed the expression of BCL11A mRNA and protein as compared with the control group. MDA-MB-231 cells transfected with BCL11A-siRNA had higher apoptosis rate compared with the control group. Conclusion The BCL11A protein is highly expressed in breast cancer and knock-down of BCL11A promotes the apoptosis of MDA-MB-231 cells.

Copper-64-labeled anti-bcl-2 PNA-peptide conjugates selectively localize to bcl-2-positive tumors in mouse models of B-cell lymphoma.

PubMed

Jia, Fang; Balaji, Baghavathy S; Gallazzi, Fabio; Lewis, Michael R

2015-11-01

The bcl-2 gene is overexpressed in non-Hodgkin's lymphoma (NHL). We have reported micro-SPECT/CT imaging of Mec-1 human lymphoma xenografts in SCID mice, using [(111)In]DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate. In order to reduce normal organ accumulation and improve imaging contrast, modified monomers with neutral hydrophilic (serine, TS) or negatively charged (aspartic acid, TD) residues were synthesized as substitutes for glycine at T(14) in the PNA sequence. The parent and modified PNA-peptide conjugates were labeled with (64)Cu and evaluated in biodistribution studies and high resolution PET/CT imaging of SCID mice bearing bcl-2-positive Mec-1 xenografts as well as bcl-2-negative Ramos xenografts. Mice were administered the (64)Cu-labeled conjugates for biodistribution and imaging studies. Biodistributions were obtained from 1 to 48 h post-injection. Mice were imaged from 1 to 48 h post-injection. The parent glycine conjugate and two modified conjugates all showed selective tumor uptake in Mec-1 xenografts. The liver uptake of the serine conjugate was significantly reduced compared to the two other PNA conjugates. Its kidney uptake was highest of the three at 47.1% ID/g at 1h and dropped to 20.6% ID/g at 24h. [Copper-64]DOTA-anti-bcl-2-TS-PNA-Tyr(3)-octreotate showed tumor uptake of 1.38% ID/g at 1h and 1.06% ID/g at 24h. The tumor-to-blood ratio was increased by factor of 2 from 1h to 24h. This compound detected Mec-1 tumors by micro-PET/CT as early as 1h post-injection and at time points out to 48 h. However, the negative control Ramos tumor could not be detected. These (64)Cu-labeled, amino acid-modified PNA conjugates showed selective tumor targeting in vivo, and tumor xenografts were detected by micro-PET/CT as early as 1h post-injection, suggesting that bcl-2 expression at the mRNA level can detected by PET in mouse models of NHL. Advances in knowledge and implications for patient care Down-regulating bcl-2, an anti-apoptotic proto-oncogene, is a mechanism

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Sogabe, Satoshi; Kamada, Yusuke

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, K{sub D} and IC{sub 50} values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibitedmore » relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported. - Highlights: • F1324 was discovered as 5000-times higher affinity peptide to BCL6 than that of BcoR(R498-P514). • X-ray crystal structure analysis revealed the binding mode. • To our knowledge, F1324 is the strongest BCL6-binding and -inhibition peptide so far.« less

Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia

PubMed Central

Li, Sheng-Mian; Yao, Shu-Kun; Yamamura, Nobuyoshi; Nakamura, Toshitsugu

2003-01-01

AIM: To compare the difference of expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia, and to analyze the role of Bcl-2 and Bax proteins in the progression from dysplasia to carcinoma and to evaluate the correlation of Bcl-2/Bax protein expression with the biological behaviors. METHODS: Expressions of Bcl-2 and Bax were examined immunohistochemically in 27 cases of extrahepatic biliary tract carcinomas (bile duct carcinoma: n = 21, carcinoma of ampulla of Vater: n = 6), and 10 cases of atypical dysplasia. Five cases of normal biliary epithelial tissues were used as controls. A semiquantitative scoring system was used to assess the Bcl-2 and Bax reactivity. RESULTS: The expression of Bcl-2 was observed in 10 out of 27 (37.0%) invasive carcinomas, 1 out of 10 dysplasias, none out of 5 normal epithelial tissues. Bax expression rate was 74.1% (20/27) in invasive carcinoma, 30% (3/10) in dysplasia, and 40% (2/5) in normal biliary epithelium. Bcl-2 and Bax activities were more intense in carcinoma than in dysplasia, with no significant difference in Bcl-2 expression (P = 0.110), and significant difference in Bax expression (P = 0.038). Level of Bax expression was higher in invasive carcinoma than in dysplasia and normal tissue (P = 0.012). Bcl-2 expression was correlated to Bax expression (P = 0.0059). However, Bcl-2/Bax expression had no correlation with histological subtype, grade of differentiation, or level of invasion. CONCLUSION: Increased Bcl-2/Bax expression from dysplasia to invasive tumors supports the view that this is the usual route for the development of extrahepatic biliary tract carcinoma. Bcl-2/Bax may be involved, at least in part, in the apoptotic activity in extrahepatic biliary carcinoma. PMID:14606101

Development of chronic allergic responses by dampening Bcl6-mediated suppressor activity in memory T helper 2 cells

PubMed Central

Ogasawara, Takashi; Hatano, Masahiko; Satake, Hisae; Ikari, Jun; Taniguchi, Toshibumi; Tsuruoka, Nobuhide; Watanabe-Takano, Haruko; Fujimura, Lisa; Sakamoto, Akemi; Hirata, Hirokuni; Sugiyama, Kumiya; Fukushima, Yasutsugu; Nakae, Susumu; Matsumoto, Kenji; Saito, Hirohisa; Fukuda, Takeshi; Kurasawa, Kazuhiro; Tatsumi, Koichiro; Tokuhisa, Takeshi

2017-01-01

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularly Interleukin4 (Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, including Il4 loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6’s association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation. PMID:28096407

Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats

PubMed Central

Qiao, Wei-Li; Wang, Guang-Ming; Shi, Yue; Wu, Jin-Xia; Qi, You-Jian; Zhang, Jian-Fu; Sun, Hong; Yan, Chang-Dong

2011-01-01

AIM: To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation. METHODS: The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in Sprague-Dawley rats. Rats were assigned to groups in accordance with their evaluation period: control, 0, 0.5, 1, 3, 6, 24, 48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice. RESULTS: Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased in the early phases of reperfusion, reached its minimum at 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expression increased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05). On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor, had significant effects on Bcl-2 and Bax in GI-R. Compared with GI-R treatment only at 3 h of reperfusion, PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 protein level (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P < 0.05) and Bax protein level (1.35-fold of R3h group, P < 0.05). CONCLUSION: These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2. PMID:21483632

Bupropion XL-induced motor and vocal tics.

PubMed

Kayhan, Fatih; Uguz, Faruk; Kayhan, Ayşegül; Toktaş, Fikriye Ilay

2014-01-01

Tics are stereotypical repetitive involuntary movements (motor tics) or sounds (vocal tics). Although the emergence of tics were reported in a few cases with the use of selective serotonin reuptake inhibitors, there was no case with bupropion extended-release (Bupropion XL). The current case report presents a male patient developing motor and vocal tics with the use of bupropion XL.

Targeting Bcl-2 stability to sensitize cells harboring oncogenic ras.

PubMed

Peng, Bo; Ganapathy, Suthakar; Shen, Ling; Huang, Junchi; Yi, Bo; Zhou, Xiaodong; Dai, Wei; Chen, Changyan

2015-09-08

The pro-survival factor Bcl-2 and its family members are critical determinants of the threshold of the susceptibility of cells to apoptosis. Studies are shown that cells harboring an oncogenic ras were extremely sensitive to the inhibition of protein kinase C (PKC) and Bcl-2 could antagonize this apoptotic process. However, it remains unrevealed how Bcl-2 is being regulated in this apoptotic process. In this study, we investigate the role of Bcl-2 stability in sensitizing the cells harboring oncogenic K-ras to apoptosis triggered by PKC inhibitor GO6976. We demonstrated that Bcl-2 in Swiss3T3 cells ectopically expressing or murine lung cancer LKR cells harboring K-ras rapidly underwent ubiquitin-dependent proteasome pathway after the treatment of GO6976, accompanied with induction of apoptosis. In this process, Bcl-2 formed the complex with Keap-1 and Cul3. The mutation of serine-17 and deletion of BH-2 or 4 was required for Bcl-2 ubiquitination and degradation, which elevate the signal threshold for the induction of apoptosis in the cells following PKC inhibition. Thus, Bcl-2 appears an attractive target for the induction of apoptosis by PKC inhibition in cancer cells expressing oncogenic K-ras.

The BCL-2 family protein Bid is critical for pro-inflammatory signaling in astrocytes.

PubMed

König, Hans-Georg; Coughlan, Karen S; Kinsella, Sinéad; Breen, Bridget A; Prehn, Jochen H M

2014-10-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1(G93A) transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1(G93A) mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Pegasus XL CYGNSS Fairing Mate Complete

NASA Image and Video Library

2016-11-15

In Building 1555 at Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is seen after payload fairing installation. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

Identification of the Microtubule-Inhibitor Activated Bcl-xL Kinase: A Regulator of Breast Cancer Cell Chemosensitivity to Taxol

DTIC Science & Technology

2011-08-01

not shown). 1 5 Translational research study We continued the clinical protocol entitled “Alterations in the Bcl-2 Family Proteins...Bcl- 2 protein phosphorylation in hematopoietic cells may be more likely to have fever, neutropenia than others, for example. While effective...Alterations in the Bcl-2 Family Proteins in the Peripheral Blood Following Treatment with Taxanes in Patients with Breast Cancer.” Accepted by the UAMS

Discovery and molecular characterization of a Bcl-2–regulated cell death pathway in schistosomes

PubMed Central

Lee, Erinna F.; Clarke, Oliver B.; Evangelista, Marco; Feng, Zhiping; Speed, Terence P.; Tchoubrieva, Elissaveta B.; Strasser, Andreas; Kalinna, Bernd H.; Colman, Peter M.; Fairlie, W. Douglas

2011-01-01

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2–regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2–like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with “BH3 mimetic” drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment. PMID:21444803

A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

PubMed

Lavik, Andrew R; Zhong, Fei; Chang, Ming-Jin; Greenberg, Edward; Choudhary, Yuvraj; Smith, Mitchell R; McColl, Karen S; Pink, John; Reu, Frederic J; Matsuyama, Shigemi; Distelhorst, Clark W

2015-09-29

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

Expression of apoptosis related proteins: RAIDD, ZIP kinase, Bim/BOD, p21, Bax, Bcl-2 and NF-kappaB in brains of patients with Down syndrome.

PubMed

Engidawork, E; Gulesserian, T; Seidl, R; Cairns, N; Lubec, G

2001-01-01

Down syndrome (DS) is a genetic disease that exhibits significant neuropathological parallels with Alzheimer's disease (AD). One of the features of DS, neuronal loss, has been hypothesized to occur as a result of apoptosis. An increasing number of proteins are implicated in apoptosis and several of them were shown to be altered in AD, however, the knowledge in DS is far from complete. To further substantiate the hypothesis that apoptosis is the underlying mechanism for neuronal loss and contribute towards the current knowledge of apoptosis in DS, we analyzed the expression of apoptosis related proteins in frontal cortex and cerebellum of DS by western blot and ELISA techniques. Quantitative analysis revealed a significant increase in DS frontal (P < 0.0001) and cerebellar (P < 0.05) Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene), cerebellar Bcl-2 (P < 0.01) as well as p21 (P < 0.05) levels compared to controls. No significant change was detected in Bax, RAIDD (receptor interacting protein (RIP)-associated ICH-1/CED-3-homologus protein with death domain), ZIP (Zipper interacting protein) kinase and NF-kappaB p65 levels in both regions, although frontal cortex levels of RAIDD, Bcl-2 and p21 levels tended to increase. In addition, a 45 kDa truncated form of NF-kappaB p65 displayed a significant elevation (P < 0.05) in DS cerebellum. No significant correlation had been obtained between postmortem interval and level of the proteins analyzed. With regard to age, it was only NF-kappaB p65 that showed significant correlation (r = -0.8964, P = 0.0155, n = 9) in frontal cortex of controls. These findings provide further evidence that apoptosis indeed accounts for the neuronal loss in DS but Bax and RAIDD do not appear to take part in this process.

Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

PubMed Central

Marshall, B; Puthalakath, H; Caria, S; Chugh, S; Doerflinger, M; Colman, P M; Kvansakul, M

2015-01-01

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism. PMID:25766319

Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim.

PubMed

Marshall, B; Puthalakath, H; Caria, S; Chugh, S; Doerflinger, M; Colman, P M; Kvansakul, M

2015-03-12

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth.

PubMed

Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H; Galipeau, Jacques; Deng, Xingming

2016-05-10

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

ABT-199 (venetoclax) and BCL-2 inhibitors in clinical development.

PubMed

Cang, Shundong; Iragavarapu, Chaitanya; Savooji, John; Song, Yongping; Liu, Delong

2015-11-20

With the advent of new agents targeting CD20, Bruton's tyrosine kinase, and phosphoinositol-3 kinase for chronic lymphoid leukemia (CLL), more treatment options exist than ever before. B-cell lymphoma-2 (BCL-2) plays a major role in cellular apoptosis and is a druggable target. Small molecule inhibitors of BCL-2 are in active clinical studies. ABT-199 (venetoclax, RG7601, GDC-0199) has been granted breakthrough designation by FDA for relapsed or refractory CLL with 17p deletion. In this review, we summarized the latest clinical development of ABT-199/venetoclax and other novel agents targeting the BCL-2 proteins.

New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463.

PubMed

Zhu, Yi; Doornebal, Ewald J; Pirtskhalava, Tamar; Giorgadze, Nino; Wentworth, Mark; Fuhrmann-Stroissnigg, Heike; Niedernhofer, Laura J; Robbins, Paul D; Tchkonia, Tamara; Kirkland, James L

2017-03-08

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-X L inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

Pegasus XL CYGNSS

NASA Image and Video Library

2016-09-15

Inside Building 1555 at Vandenberg Air Force Base in California, technicians and engineers install the first stage aft skirt on the Orbital ATK Pegasus XL rocket which will launch eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are completed at Vandenberg, the rocket, with CYGNSS in its payload fairing, will be attached to the Orbital ATK L-1011 carrier aircraft and transported to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Antagonism between MCL-1 and PUMA governs stem/progenitor cell survival during hematopoietic recovery from stress

PubMed Central

Delbridge, Alex R. D.; Opferman, Joseph T.; Grabow, Stephanie

2015-01-01

Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma–extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1+/−;Puma−/− mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress. PMID:25847014

The study of the intercellular trafficking of the fusion proteins of herpes simplex virus protein VP22.

PubMed

Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

2014-01-01

Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP22-hBcl-xL and hBcl-xL-VP22 failed to

The Study of the Intercellular Trafficking of the Fusion Proteins of Herpes Simplex Virus Protein VP22

PubMed Central

Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

2014-01-01

Background Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Methods Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. Results VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. Conclusions The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP

BCL-2 system analysis identifies high-risk colorectal cancer patients.

PubMed

Lindner, Andreas U; Salvucci, Manuela; Morgan, Clare; Monsefi, Naser; Resler, Alexa J; Cremona, Mattia; Curry, Sarah; Toomey, Sinead; O'Byrne, Robert; Bacon, Orna; Stühler, Michael; Flanagan, Lorna; Wilson, Richard; Johnston, Patrick G; Salto-Tellez, Manuel; Camilleri-Broët, Sophie; McNamara, Deborah A; Kay, Elaine W; Hennessy, Bryan T; Laurent-Puig, Pierre; Van Schaeybroeck, Sandra; Prehn, Jochen H M

2017-12-01

The mitochondrial apoptosis pathway is controlled by an interaction of multiple BCL-2 family proteins, and plays a key role in tumour progression and therapy responses. We assessed the prognostic potential of an experimentally validated, mathematical model of BCL-2 protein interactions (DR_MOMP) in patients with stage III colorectal cancer (CRC). Absolute protein levels of BCL-2 family proteins were determined in primary CRC tumours collected from n=128 resected and chemotherapy-treated patients with stage III CRC. We applied DR_MOMP to categorise patients as high or low risk based on model outputs, and compared model outputs with known prognostic factors (T-stage, N-stage, lymphovascular invasion). DR_MOMP signatures were validated on protein of n=156 patients with CRC from the Cancer Genome Atlas (TCGA) project. High-risk stage III patients identified by DR_MOMP had an approximately fivefold increased risk of death compared with patients identified as low risk (HR 5.2, 95% CI 1.4 to 17.9, p=0.02). The DR_MOMP signature ranked highest among all molecular and pathological features analysed. The prognostic signature was validated in the TCGA colon adenocarcinoma (COAD) cohort (HR 4.2, 95% CI 1.1 to 15.6, p=0.04). DR_MOMP also further stratified patients identified by supervised gene expression risk scores into low-risk and high-risk categories. BCL-2-dependent signalling critically contributed to treatment responses in consensus molecular subtypes 1 and 3, linking for the first time specific molecular subtypes to apoptosis signalling. DR_MOMP delivers a system-based biomarker with significant potential as a prognostic tool for stage III CRC that significantly improves established histopathological risk factors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Pegasus XL CYGNSS Move to AIT

NASA Image and Video Library

2016-11-17

At Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is placed on an assembly integration transporter for the move from the hangar at Building 1555 to be mated to L-1011 carrier aircraft near Vandenberg's runway. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

PubMed

Akl, Haidar; Vervloessem, Tamara; Kiviluoto, Santeri; Bittremieux, Mart; Parys, Jan B; De Smedt, Humbert; Bultynck, Geert

2014-10-01

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. Copyright © 2014 Elsevier B.V. All rights

MYC/BCL2/BCL6 triple hit lymphoma: a study of 40 patients with a comparison to MYC/BCL2 and MYC/BCL6 double hit lymphomas.

PubMed

Huang, Wenting; Medeiros, L Jeffrey; Lin, Pei; Wang, Wei; Tang, Guilin; Khoury, Joseph; Konoplev, Sergej; Yin, C Cameron; Xu, Jie; Oki, Yasuhiro; Li, Shaoying

2018-05-21

High-grade B-cell lymphomas with MYC, BCL2, and BCL6 rearrangements (triple hit lymphoma) are uncommon. We studied the clinicopathologic features of 40 patients with triple hit lymphoma and compared them to 157 patients with MYC/BCL2 double hit lymphoma and 13 patients with MYC/BCL6 double hit lymphoma. The triple hit lymphoma group included 25 men and 15 women with a median age of 61 years (range, 34-85). Nine patients had a history of B-cell lymphoma. Histologically, 23 (58%) cases were diffuse large B-cell lymphoma and 17 cases had features of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. Most cases of triple hit lymphoma were positive for CD10 (100%), BCL2 (95%), BCL6 (82%), MYC (74%), and 71% with MYC and BCL2 coexpression. P53 was overexpressed in 29% of triple hit lymphoma cases. The clinicopathological features of triple hit lymphoma patients were similar to patients with MYC/BCL2 and MYC/BCL6 double hit lymphoma, except that triple hit lymphoma cases were more often CD10 positive compared with MYC/BCL6 double hit lymphoma (p < 0.05). Induction chemotherapy used was similar for patients with triple hit lymphoma and double hit lymphoma and overall survival in triple hit lymphoma patients was 17.6 months, similar to the overall survival of patients with double hit lymphoma (p = 0.67). Patients with triple hit lymphoma showing P53 overexpression had significantly worse overall survival compared with those without P53 overexpression (p = 0.04). On the other hand, double expressor status and prior history of B-cell lymphoma did not correlate with overall survival. In conclusion, most patients with triple hit lymphoma have an aggressive clinical course and poor prognosis and these tumors have a germinal center B-cell immunophenotype, similar to patients with double hit lymphomas. P53 expression is a poor prognostic factor in patients with triple hit lymphoma.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

Bcl-2 protein expression associated with resistance to apoptosis in clear cell adenocarcinomas of the vagina and cervix expressing wild-type p53.

PubMed

Waggoner, S E; Baunoch, D A; Anderson, S A; Leigh, F; Zagaja, V G

1998-09-01

Clear cell adenocarcinomas (CCAs) of the vagina and cervix are rare tumors that often overexpress wild-type p53. In vitro, expression of protooncogene bcl-2 can block p53-mediated apoptosis. The objective of this study was to determine if bcl-2 is expressed in CCAs and whether this expression is associated with inhibition of apoptosis. Twenty-one paraffin-embedded clear cell adenocarcinomas were immunohistochemically stained for bcl-2 (antibody M 887, Dako, Carpinteria, CA) and DNA fragmentation (ApopTag, Oncor, Gaithersburg, MD), a marker for apoptosis. Fifteen tumors were associated with in utero exposure to diethylstilbestrol (DES). Prior p53 gene analysis had indicated the presence of wild-type p53 in each tumor. Human lymphoid tissue containing bcl-2-expressing lymphocytes and DNase I-exposed CCA tissue sections were used as positive controls for the bcl-2 and apoptosis assays, respectively. Expression of bcl-2 and DNA fragmentation was classified (0 to 3+) according to percentage of positive cells and intensity of staining. Expression of bcl-2 was identified in each CCA examined, and was strongly positive (2+ to 3+) in 18 of 21 samples. Despite the presence of wild-type p53, only 4 of 21 tumors showed evidence of apoptosis as assessed through DNA fragmentation. DNA damage leads to increased intracellular p53 levels. Overexpression of p53 induces apoptosis as a means of protecting organisms from the development of malignancy. CCAs of the vagina and cervix, which contain wild-type p53 genes and often overexpress p53 protein, presumably have evolved mechanisms to avoid p53-induced apoptosis. Our observations are consistent with the hypothesis that overexpression of bcl-2 can inhibit p53-mediated apoptosis and suggest a mechanism by which these rare tumors can arise without mutation of the p53 gene.

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

PubMed Central

Malina, Halina Z; Hess, Otto M

2004-01-01

Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions. PMID:15068490

Inherited BCL10 deficiency impairs hematopoietic and nonhematopoietic immunity

PubMed Central

Torres, Juan Manuel; Martinez-Barricarte, Rubén; García-Gómez, Sonia; Mazariegos, Marina S.; Itan, Yuval; Boisson, Bertrand; ρlvarez, Rita; Jiménez-Reinoso, Anaïs; del Pino, Lucia; Rodríguez-Pena, Rebeca; Ferreira, Antonio; Hernández-Jiménez, Enrique; Toledano, Victor; Cubillos-Zapata, Carolina; Díaz-Almirón, Mariana; López-Collazo, Eduardo; Unzueta-Roch, José L.; Sánchez-Ramón, Silvia; Regueiro, Jose R.; López-Granados, Eduardo; Casanova, Jean-Laurent; Pérez de Diego, Rebeca

2014-01-01

Heterotrimers composed of B cell CLL/lymphoma 10 (BCL10), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and caspase recruitment domain–containing (CARD) family adaptors play a role in NF-κB activation and have been shown to be involved in both the innate and the adaptive arms of immunity in murine models. Moreover, individuals with inherited defects of MALT1, CARD9, and CARD11 present with immunological and clinical phenotypes. Here, we characterized a case of autosomal-recessive, complete BCL10 deficiency in a child with a broad immunodeficiency, including defects of both hematopoietic and nonhematopoietic immunity. The patient died at 3 years of age and was homozygous for a loss-of-expression, loss-of-function BCL10 mutation. The effect of BCL10 deficiency was dependent on the signaling pathway, and, for some pathways, the cell type affected. Despite the noted similarities to BCL10 deficiency in mice, including a deficient adaptive immune response, human BCL10 deficiency in this patient resulted in a number of specific features within cell populations. Treatment of the patient’s myeloid cells with a variety of pathogen-associated molecular pattern molecules (PAMPs) elicited a normal response; however, NF-κB–mediated fibroblast functions were dramatically impaired. The results of this study indicate that inherited BCL10 deficiency should be considered in patients with combined immunodeficiency with B cell, T cell, and fibroblast defects. PMID:25365219

The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

PubMed Central

Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

2013-01-01

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

PubMed

Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

2013-03-08

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer.

PubMed

Veit, Johannes; Sachsenberg, Timo; Chernev, Aleksandar; Aicheler, Fabian; Urlaub, Henning; Kohlbacher, Oliver

2016-09-02

Modern mass spectrometry setups used in today's proteomics studies generate vast amounts of raw data, calling for highly efficient data processing and analysis tools. Software for analyzing these data is either monolithic (easy to use, but sometimes too rigid) or workflow-driven (easy to customize, but sometimes complex). Thermo Proteome Discoverer (PD) is a powerful software for workflow-driven data analysis in proteomics which, in our eyes, achieves a good trade-off between flexibility and usability. Here, we present two open-source plugins for PD providing additional functionality: LFQProfiler for label-free quantification of peptides and proteins, and RNP(xl) for UV-induced peptide-RNA cross-linking data analysis. LFQProfiler interacts with existing PD nodes for peptide identification and validation and takes care of the entire quantitative part of the workflow. We show that it performs at least on par with other state-of-the-art software solutions for label-free quantification in a recently published benchmark ( Ramus, C.; J. Proteomics 2016 , 132 , 51 - 62 ). The second workflow, RNP(xl), represents the first software solution to date for identification of peptide-RNA cross-links including automatic localization of the cross-links at amino acid resolution and localization scoring. It comes with a customized integrated cross-link fragment spectrum viewer for convenient manual inspection and validation of the results.

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

PubMed

Schelman, William R; Andres, Robert D; Sipe, Kimberly J; Kang, Evan; Weyhenmeyer, James A

2004-09-28

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of

Pegasus XL CYGNSS Second Launch Attempt

NASA Image and Video Library

2016-12-15

An Orbital ATK L-1011 Stargazer aircraft carrying a Pegasus XL Rocket with eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, soars high after takeoff from the Skid Strip at Cape Canaveral Air Force Station, Florida. With the aircraft flying off shore, the Pegasus rocket will be released. Five seconds later, the solid propellant engine will ignite and boost the eight hurricane observatories to orbit. The eight CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. Release of the Pegasus XL rocket is scheduled for 8:40 a.m. EST.

Pegasus XL CYGNSS Second Launch Attempt

NASA Image and Video Library

2016-12-15

An Orbital ATK L-1011 Stargazer aircraft descends toward the Skid Strip at Cape Canaveral Air Force Station in Florida. The aircraft carried a Pegasus XL Rocket with eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, for launch. With the aircraft flying off shore, the Pegasus rocket was released. Five seconds later, the solid propellant engine ignited and boosted the eight hurricane observatories to orbit. The eight CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. Release of the Pegasus XL rocket occurred at 8:37 a.m. EST.

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

PubMed

Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

2013-12-01

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1

Pegasus XL CYGNSS - "Days to Launch" Sign

NASA Image and Video Library

2016-12-05

A sign just inside the gate to NASA's Kennedy Space Center in Florida notes that in seven days a Pegasus XL rocket is scheduled to launch with eight agency Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. On Dec. 12, 2016, the Orbital ATK L-1011 Stargazer, with a Pegasus XL rocket mated to the underside of the aircraft, will take off from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will help scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a crucial role in the beginning and intensification of hurricanes.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth

PubMed Central

Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H.; Galipeau, Jacques; Deng, Xingming

2016-01-01

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy. PMID:27049723

Bcl-2 protects tubular epithelial cells from ischemia/reperfusion injury by dual mechanisms.

PubMed

Isaka, Y; Suzuki, C; Abe, T; Okumi, M; Ichimaru, N; Imamura, R; Kakuta, Y; Matsui, I; Takabatake, Y; Rakugi, H; Shimizu, S; Takahara, S

2009-01-01

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.

Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models.

PubMed

Tan, Nguyen; Wong, Maureen; Nannini, Michelle A; Hong, Rebecca; Lee, Leslie B; Price, Stephen; Williams, Karen; Savy, Pierre Pascal; Yue, Peng; Sampath, Deepak; Settleman, Jeffrey; Fairbrother, Wayne J; Belmont, Lisa D

2013-06-01

Although mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non-small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. ©2013 AACR

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis.

PubMed

Weber, Stefan U; Schewe, Jens-Christian; Lehmann, Lutz E; Müller, Stefan; Book, Malte; Klaschik, Sven; Hoeft, Andreas; Stüber, Frank

2008-01-01

In transgenic animal models of sepsis, members of the Bcl-2 family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins in patients with early stage severe sepsis. In this prospective case-control study, patients were recruited from three intensive care units (ICUs) in a university hospital. Sixteen patients were enrolled when they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and 11 healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine externalisation in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed using flow cytometry. Specific mRNAs of Bcl-2 family members were quantified from whole blood by real-time PCR. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc analysis was performed. In all lymphocyte populations caspase-3 (p < 0.05) was activated, which was reflected in an increased phosphatidylserine externalisation (p < 0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p < 0.05) and B-cells (p < 0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared with critically ill patients (p < 0.001) and 51.6-fold as compared with healthy controls (p < 0.05). Bid was increased 12.9-fold compared with critically ill patients (p < 0.001). In the group of mitochondrial apoptosis inducers, Bak was upregulated 5.6-fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p < 0.001 and p < 0.05, respectively). In early severe sepsis a gene expression pattern with

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 overexpressing radioresistant tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara, Takamitsu; Omura-Minamisawa, Motoko; Chao Cheng

Purpose: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. Methods and materials: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cellmore » viability was then determined. Apoptotic cells were quantified by flow cytometric assay. Results: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. Conclusions: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2.« less

Clinicopathological correlations of Bcl-xL and Bax expression in differentiated thyroid carcinoma.

PubMed

Martínez-Brocca, M Asunción; Castilla, Carolina; Navarro, Elena; Amaya, M José; Travado, Paulino; Japón, Miguel A; Sáez, Carmen

2008-02-01

The Bcl-2 family proteins are essential mediators in the apoptotic process. Our aim was to investigate whether anti-apoptotic Bcl-xL and pro-apoptotic Bax were over-expressed in a large series of differentiated thyroid carcinomas (DTC) and to study their association with tumour presentation at diagnosis and prognosis. We examined the immunohistochemical expression of Bcl-xL and Bax in benign nodular thyroid disease (BNTD) and DTC and their association with clinicopathological parameters. Thyroid tissue samples were collected from an unselected series of patients undergoing surgical resection for DTC (n = 74) or BNTD (n = 15). Among DTC cases, expression of Bcl-xL was found to be high in 43.2% and low or absent in 56.8%. Expression of Bax was high in 75.7% and low or absent in 24.3%. Non-neoplastic thyroid tissue was largely unstained for both proteins. Among BNTD cases, expression of Bcl-xL was high in 13.3% and low or absent in 86.6%. Expression of Bax was high in 14.3% and low or absent in 86.6%. A significant association was found between Bcl-xL expression and the presence of high-risk histological subtype (P < 0.05), and regional lymph node (P < 0.01) and distant metastases (P < 0.01). The association between high Bcl-xL expression levels and a longer time of persistent disease after radioiodine ablation was also significant (P < 0.01). Bcl-xL expression was confirmed as an independent prognostic factor for persistent disease in DTC (relative risk, 2.5; 95% confidence interval, 1.1-5.9; P < 0.05). Immunohistochemical expression of Bcl-xL might be a valuable tool in the prediction of tumour aggressiveness in DTC.

Hierarchy for targeting prosurvival BCL2 family proteins in multiple myeloma: pivotal role of MCL1.

PubMed

Gong, Jia-Nan; Khong, Tiffany; Segal, David; Yao, Yuan; Riffkin, Chris D; Garnier, Jean-Marc; Khaw, Seong Lin; Lessene, Guillaume; Spencer, Andrew; Herold, Marco J; Roberts, Andrew W; Huang, David C S

2016-10-06

New therapeutic targets are needed to address the poor prognosis of patients with high-risk multiple myeloma. Myeloma cells usually express a range of the prosurvival BCL2 proteins. To define the hierarchy of their relative importance for maintaining the survival of myeloma cells, we targeted each of them in a large panel of cell lines, using pharmacological inhibitors or gene editing or by peptide-based approaches, alone or in combination. The majority of well-established immortalized cell lines (17/25) or low-passage myeloma cell lines (5/7) are readily killed when MCL1 is targeted, even including those cell lines sensitive to BCL2 inhibition. Targeting MCL1 also constrained the growth of myeloma in vivo. We also identified a previously unrecognized subset of myeloma that is highly BCLXL-dependent, and has the potential for cotargeting MCL1 and BCLXL. As MCL1 is pivotal for maintaining survival of most myelomas, it should be prioritized for targeting in the clinic once high-quality, validated inhibitors become available. © 2016 by The American Society of Hematology.

Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

PubMed

He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

2016-01-01

Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better

Complex disruption effect of natural polyphenols on Bcl-2-Bax: molecular dynamics simulation and essential dynamics study.

PubMed

Verma, Sharad; Singh, Amit; Mishra, Abha

2015-01-01

Apoptosis (programmed cell death) is a process by which cells died after completing physiological function or after a severe genetic damage. Apoptosis is mainly regulated by the Bcl-2 family of proteins. Anti apoptotic protein Bcl-2 prevents the Bax activation/oligomerization to form heterodimer which is responsible for release of the cytochrome c from mitochondria to the cytosol in response to death signal. Quercetin and taxifolin (natural polyphenols) efficiently bound to hydrophobic groove of Bcl-2 and altered the structure by inducing conformational changes. Taxifolin was found more efficient when compared to quercetin in terms of interaction energy and collapse of hydrophobic groove. Taxifolin and quercetin were found to dissociate the Bcl-2-Bax complex during 12 ns MD simulation. The effect of taxifolin and quercetin was, further validated by the MD simulation of ligand-unbound Bcl-2-Bax which showed stability during the simulation. Obatoclax (an inhibitor of Bcl-2) had no significant dissociation effect on Bcl-2-Bax during simulation which favored the previous experimental results and disruption effect of taxifolin and quercetin.

B-cell transcription factors Pax-5, Oct-2, BOB.1, Bcl-6, and MUM1 are useful markers for the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma.

PubMed

Herbeck, Rosemarie; Teodorescu Brînzeu, D; Giubelan, Marioara; Lazăr, Elena; Dema, Alis; Ioniţă, Hortensia

2011-01-01

In some instances, the overlap in morphologic features and antigen expression between nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (cHL) can cause confusion in the diagnosis. In these cases, the transcription factors (TFs) B-cell specific activator protein (BSAP)/Pax-5, octamer binding protein-2 (Oct-2), B-lymphocyte-specific co-activator BOB.1/OBF.1, Bcl-6 protein and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF-4) may aid in clarifying the diagnosis. Twenty-two cases of NLPHL were studied for the immunohistochemical expression of Pax-5, Oct-2, BOB.1, Bcl-6 protein and MUM1/IRF-4. Our results sustain the usefulness of the selected set of TFs to diagnose and distinguish NLPHL from cHL since Pax-5, Oct-2, BOB.1 and Bcl-6 are consistently expressed by lymphocyte predominant (LP) cells and reported by others to be often unexpressed in Hodgkin and Reed-Sternberg cells. By contrast, MUM1/IRF-4 protein scored negative in the majority of LP cells, but is reported to be expressed in almost all cases of cHL. Thus, although the expression of transcription factors is very heterogeneous, their simultaneous implementation for positive and differential diagnosis may be useful.

Stress Hormone Cortisol Enhances Bcl2 Like-12 Expression to Inhibit p53 in Hepatocellular Carcinoma Cells.

PubMed

Wu, Weizhong; Liu, Sanguang; Liang, Yunfei; Zhou, Zegao; Bian, Wei; Liu, Xueqing

2017-12-01

The pathogenesis of hepatocellular carcinoma (HC) is unclear. It is suggested that psychological stress associates with the pathogenesis of liver cancer. Bcl2-like protein 12 (Bcl2L12) suppresses p53 protein. This study tests a hypothesis that the major stress hormone, cortisol, inhibits the expression of p53 in HC cells (HCC) via up regulating the expression of Bcl2L12. Peripheral blood samples were collected from patients with HC to be analyzed for the levels of cortisol. HCC were cultured to assess the role of cortisol in the regulation of the expression of Bcl2L12 and p53 in HCC. We observed that the serum cortisol levels were higher in HC patients. Expression of Bcl2L12 in HCC was correlated with serum cortisol. Cortisol enhanced the Bcl2L12 expression in HCC. Bcl2L12 binding to the TP53 promoter was correlated with p53 expression in HCC. Cortisol increased the Bcl2L12 expression in HCC to inhibit p53 expression. Stress hormone cortisol suppresses p53 in HCC via enhancing Bcl2L12 expression in HCC. The results suggest that cortisol may be a therapeutic target for the treatment of HC.

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Cui-Li, E-mail: zhangcuili@hotmail.com; Song, Fei; Zhang, Jing

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580)more » blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs' proliferation and migration. Over-expression of Bcl-2 increased HAECs' tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.« less

Pegasus XL CYGNSS Mate to L-1011

NASA Image and Video Library

2016-11-28

At Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is transported to be mated to the company's L-1011 carrier aircraft near Vandenberg's runway. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Mate to L-1011

NASA Image and Video Library

2016-11-28

At Vandenberg Air Force Base in California, an Orbital ATK Pegasus XL rocket is mated to the company's L-1011 carrier aircraft near Vandenberg's runway. On board Pegasus are eight NASA Cyclone Global Navigation Satellite System, or CYGNSS, spacecraft. When preparations are competed at Vandenberg, the L-1011/Pegasus XL combination will be flown to NASA’s Kennedy Space Center in Florida. On Dec. 12, 2016, the carrier aircraft is scheduled to take off from the Skid Strip at Cape Canaveral Air Force Station and CYGNSS will launch on the Pegasus XL rocket with the L-1011 flying off shore. CYGNSS satellites will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

PubMed

Ochiai, Kyoko; Muto, Akihiko; Tanaka, Hiromu; Takahashi, Shinichiro; Igarashi, Kazuhiko

2008-03-01

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

Lifesource XL-18 pedometer for measuring steps under controlled and free-living conditions.

PubMed

Liu, Sam; Brooks, Dina; Thomas, Scott; Eysenbach, Gunther; Nolan, Robert Peter

2015-01-01

The primary aim was to examine the criterion and construct validity and test-retest reliability of the Lifesource XL-18 pedometer (A&D Medical, Toronto, ON, Canada) for measuring steps under controlled and free-living activities. The influence of body mass index, waist size and walking speed on the criterion validity of XL-18 was also explored. Forty adults (35-74 years) performed a 6-min walk test in the controlled condition, and the criterion validity of XL-18 was assessed by comparing it to steps counted manually. Thirty-five adults participated in the free-living condition and the construct validity of XL-18 was assessed by comparing it to Yamax SW-200 (YAMAX Health & Sports, Inc., San Antonio, TX, USA). During the controlled condition, XL-18 did not significantly differ from criterion (P > 0.05) and no systematic error was found using Bland-Altman analysis. The accuracy of XL-18 decreased with slower walking speed (P = 0.001). During the free-living condition, Bland-Altman analysis revealed that XL-18 overestimated daily steps by 327 ± 118 than Yamax (P = 0.004). However, the absolute percent error (APE) (6.5 ± 0.58%) was still within an acceptable range. XL-18 did not differ statistically between pant pockets. XL-18 is suitable for measuring steps in controlled and free-living conditions. However, caution may be required when interpreting the steps recorded under slower speeds and free-living conditions.

Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

PubMed Central

Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

2015-01-01

Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity.

PubMed

Yi, Woelsung; Gupta, Sanjay; Ricker, Edd; Manni, Michela; Jessberger, Rolf; Chinenov, Yurii; Molina, Henrik; Pernis, Alessandra B

2017-08-15

Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (T H ) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (T FH ) cells, is critical as aberrant T FH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1-4E-BP-eIF4E axis, secondary to aberrant assembly of a raptor-p62-TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant T FH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (T FH ) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of T FH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

NASA Astrophysics Data System (ADS)

Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

2010-03-01

The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

Bcl-3 regulates TGFβ signaling by stabilizing Smad3 during breast cancer pulmonary metastasis

PubMed Central

Chen, Xi; Cao, Xinwei; Sun, Xiaohua; Lei, Rong; Chen, Pengfei; Zhao, Yongxu; Jiang, Yuhang; Yin, Jie; Chen, Ran; Ye, Deji; Wang, Qi; Liu, Zhanjie; Liu, Sanhong; Cheng, Chunyan; Mao, Jie; Hou, Yingyong; Wang, Mingliang; Siebenlist, Ulrich; Eugene Chin, Y; Wang, Ying; Cao, Liu; Hu, Guohong; Zhang, Xiaoren

2016-01-01

Transforming growth factor beta (TGFβ) signaling in breast cancer is selectively associated with pulmonary metastasis. However, the underlying mechanisms remain unclear. Here we show that Bcl-3, a member of the IκB family, serves as a critical regulator in TGFβ signaling to modulate breast cancer pulmonary metastasis. Bcl-3 expression was significantly associated with metastasis-free survival in breast cancer patients. Bcl-3 deletion inhibited the migration and invasion of breast cancer cells in vitro, as well as breast cancer lung metastasis in vivo. Bcl-3 was required for the expression of downstream TGFβ signaling genes that are involved in breast cancer lung metastasis. Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFβ treatment. Bcl-3 could bind to Smad3 and prevent the ubiquitination and degradation of Smad3 protein. These results indicate that Bcl-3 serves as a promising target to prevent breast tumor lung metastasis. PMID:27906182

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

PubMed Central

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B.; Bultynck, Geert

2017-01-01

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain. PMID:29340082

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax.

PubMed

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

2017-12-19

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca 2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP 3 R). The cell-permeable Bcl-2/IP 3 R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca 2+ -release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC 50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP 3 R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP 3 R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca 2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca 2+ -dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca 2+ -signaling events via its BH4 domain.

Implication of transcriptional repression in compound C-induced apoptosis in cancer cells

PubMed Central

Dai, R Y; Zhao, X F; Li, J J; Chen, R; Luo, Z L; Yu, L X; Chen, S K; Zhang, C Y; Duan, C Y; Liu, Y P; Feng, C H; Xia, X M; Li, H; Fu, J; Wang, H Y

2013-01-01

Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to induce apoptosis in some types of cells. However, the underlying mechanisms remain largely unclear. Using a DNA microarray analysis, we found that the expression of many genes was downregulated upon treatment with compound C. Importantly, compound C caused transcriptional repression with the induction of p53, a well-known marker of transcriptional stress response, in several cancer cell lines. Compound C did not induce the phosphorylation of p53 but dramatically increased the protein level of p53 similar to some other transcriptional inhibitors, including 5,6-dichloro-1-β-D-ribobenzimidazole (DRB). Consistent with previous reports, we found that compound C initiated apoptotic death of cancer cells in an AMPK-independent manner. Similar to DRB and actinomycin D (ActD), two classic transcription inhibitors, compound C not only resulted in the loss of Bcl-2 and Bcl-xl protein but also induced the phosphorylation of eukaryotic initiation factor-alpha (eIF2α) on Ser51. Hence, the phosphorylation of eIF2α might be a novel marker of transcriptional inhibition. It is noteworthy that compound C-mediated apoptosis of cancer cells is correlated with decreased expression of Bcl-2 and Bcl-xl and the phosphorylation of eIF2α on Ser51. Remarkably, compound C exhibits potent anticancer activities in vivo. Taken together, our data suggest that compound C may be an attractive candidate for anticancer drug development. PMID:24157877

Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells.

PubMed

Pearce, Martin C; Gamble, John T; Kopparapu, Prasad R; O'Donnell, Edmond F; Mueller, Monica J; Jang, Hyo Sang; Greenwood, Julie A; Satterthwait, Arnold C; Tanguay, Robert L; Zhang, Xiao-Kun; Kolluri, Siva Kumar

2018-05-25

Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.

Pegasus XL CYGNSS Spacecraft Arrival

NASA Image and Video Library

2016-09-28

Parts for NASA’s Cyclone Global Navigation Satellite System (CYGNSS) arrive in shipping containers and are stacked inside Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida in the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Spacecraft Arrival

NASA Image and Video Library

2016-09-28

Parts for NASA’s Cyclone Global Navigation Satellite System (CYGNSS) arrive in a shipping container at Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida in the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fairing Inspection

NASA Image and Video Library

2016-10-20

The payload fairing for an Orbital ATK Pegasus XL rocket is inspected in Building 1555 at Vandenberg Air Force Base in California. The fairing will protect NASA's Cyclone Global Navigation Satellite System (CYGNSS) spacecraft during launch. The rocket and spacecraft are being prepared at Vandenberg, then will be attached to the Orbital ATK L-1011 carrier aircraft and transported to NASA's Kennedy Space Center in Florida. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Spacecraft Arrival

NASA Image and Video Library

2016-09-28

Parts for NASA’s Cyclone Global Navigation Satellite System (CYGNSS) arrive in shipping containers at Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida in the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

ERK1/2 and the Bcl-2 Family Proteins Mcl-1, tBid, and Bim Are Involved in Inhibition of Apoptosis During Persistent Chlamydia psittaci Infection.

PubMed

Li, Li; Wang, Chuan; Wen, Yating; Hu, Yuming; Xie, Yafeng; Xu, Man; Liang, Mingxing; Liu, Wei; Liu, Liangzhuan; Wu, Yimou

2018-04-18

Chlamydia psittaci is an obligate intracellular pathogen that can cause zoonosis. Persistent C. psittaci infection can inhibit apoptosis in host cells, thus extending their survival and enabling them to complete their growth cycle. In this study, the antiapoptotic effects of persistent C. psittaci infection, induced by treatment with IFN-γ, were found to be associated with both the death receptor and the mitochondrial pathways of apoptosis. These effects were mediated by Bcl-2 family members, as evidenced by the decreased expression of proapoptotic proteins, such as tBid and Bim. Simultaneously, the antiapoptotic protein Mcl-1 was upregulated by persistent C. psittaci infection. Increased phosphorylation of ERK1/2 was observed; however, the expression of Bad, unlike that of other proapoptotic proteins, did not seem to be involved in this process. In summary, persistent chlamydial infection exerts antiapoptotic effects through both the death receptor and the mitochondrial pathways, in a process that is regulated by the ERK1/2 and apoptotic proteins of the Bcl-2 family.

Dual inhibition of Bcl-2 and Bcl-xL strikingly enhances PI3K inhibition-induced apoptosis in human myeloid leukemia cells through a GSK3- and Bim-dependent mechanism.

PubMed

Rahmani, Mohamed; Aust, Mandy Mayo; Attkisson, Elisa; Williams, David C; Ferreira-Gonzalez, Andrea; Grant, Steven

2013-02-15

Effects of concomitant inhibition of the PI3K/AKT/mTOR pathway and Bcl-2/Bcl-xL (BCL2L1) were examined in human myeloid leukemia cells. Tetracycline-inducible Bcl-2 and Bcl-xL dual knockdown sharply increased PI3K/AKT/mTOR inhibitor lethality. Conversely, inducible knockdown or dominant-negative AKT increased, whereas constitutively active AKT reduced lethality of the Bcl-2/Bcl-xL inhibitor ABT-737. Furthermore, PI3K/mTOR inhibitors (e.g., BEZ235 and PI-103) synergistically increased ABT-737-mediated cell death in multiple leukemia cell lines and reduced colony formation in leukemic, but not normal, CD34+ cells. Notably, increased lethality was observed in four of six primary acute myelogenous leukemia (AML) specimens. Responding, but not nonresponding, samples exhibited basal AKT phosphorylation. PI3K/mTOR inhibitors markedly downregulated Mcl-1 but increased Bim binding to Bcl-2/Bcl-xL; the latter effect was abrogated by ABT-737. Combined treatment also markedly diminished Bax/Bak binding to Mcl-1, Bcl-2, or Bcl-xL. Bax, Bak, or Bim (BCL2L11) knockdown or Mcl-1 overexpression significantly diminished regimen-induced apoptosis. Interestingly, pharmacologic inhibition or short hairpin RNA knockdown of GSK3α/β significantly attenuated Mcl-1 downregulation and decreased apoptosis. In a systemic AML xenograft model, dual tetracycline-inducible knockdown of Bcl-2/Bcl-xL sharply increased BEZ235 antileukemic effects. In a subcutaneous xenograft model, BEZ235 and ABT-737 coadministration significantly diminished tumor growth, downregulated Mcl-1, activated caspases, and prolonged survival. Together, these findings suggest that antileukemic synergism between PI3K/AKT/mTOR inhibitors and BH3 mimetics involves multiple mechanisms, including Mcl-1 downregulation, release of Bim from Bcl-2/Bcl-xL as well as Bak and Bax from Mcl-1/Bcl-2/Bcl-xL, and GSK3α/β, culminating in Bax/Bak activation and apoptosis. They also argue that combining PI3K/AKT/mTOR inhibitors with BH3

Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria.

PubMed

Yang, J C; Kahn, A; Cortopassi, G

2000-10-26

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca(2+)), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

PubMed Central

Hafeez, Samra; Urooj, Mahwish; Saleem, Shamiala; Gillani, Zeeshan; Shaheen, Sumaira; Qazi, Mahmood Husain; Naseer, Muhammad Imran; Iqbal, Zafar; Ansari, Shakeel Ahmed; Haque, Absarul; Asif, Muhammad; Mir, Manzoor Ahmad; Ali, Ashraf; Pushparaj, Peter Natesan; Jamal, Mohammad Sarwar; Rasool, Mahmood

2016-01-01

This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent. PMID:26745145

Contributions of Bcl-xL to acute and long term changes in bioenergetics during neuronal plasticity.

PubMed

Jonas, Elizabeth A

2014-08-01

Mitochondria manufacture and release metabolites and manage calcium during neuronal activity and synaptic transmission, but whether long term alterations in mitochondrial function contribute to the neuronal plasticity underlying changes in organism behavior patterns is still poorly understood. Although normal neuronal plasticity may determine learning, in contrast a persistent decline in synaptic strength or neuronal excitability may portend neurite retraction and eventual somatic death. Anti-death proteins such as Bcl-xL not only provide neuroprotection at the neuronal soma during cell death stimuli, but also appear to enhance neurotransmitter release and synaptic growth and development. It is proposed that Bcl-xL performs these functions through its ability to regulate mitochondrial release of bioenergetic metabolites and calcium, and through its ability to rapidly alter mitochondrial positioning and morphology. Bcl-xL also interacts with proteins that directly alter synaptic vesicle recycling. Bcl-xL translocates acutely to sub-cellular membranes during neuronal activity to achieve changes in synaptic efficacy. After stressful stimuli, pro-apoptotic cleaved delta N Bcl-xL (ΔN Bcl-xL) induces mitochondrial ion channel activity leading to synaptic depression and this is regulated by caspase activation. During physiological states of decreased synaptic stimulation, loss of mitochondrial Bcl-xL and low level caspase activation occur prior to the onset of long term decline in synaptic efficacy. The degree to which Bcl-xL changes mitochondrial membrane permeability may control the direction of change in synaptic strength. The small molecule Bcl-xL inhibitor ABT-737 has been useful in defining the role of Bcl-xL in synaptic processes. Bcl-xL is crucial to the normal health of neurons and synapses and its malfunction may contribute to neurodegenerative disease. Copyright © 2013. Published by Elsevier B.V.

Pegasus XL CYGNSS Fairing Arrival

NASA Image and Video Library

2016-10-11

Technicians with Orbital ATK move the first half of the Pegasus payload fairing for NASA’s Cyclone Global Navigation Satellite System (CYGNSS) from into Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida aboard the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

PubMed

Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

2015-03-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

The regulatory effect of Genistein on granulosa cell in ovary of rat with PCOS through Bcl-2 and Bax signaling pathways.

PubMed

Chi, Xiao-Xing; Zhang, Tao; Chu, Xiao-Li; Zhen, Jing-Long; Zhang, Dong-Jie

2018-06-22

The effect of genistein on Bcl-2 and Bax protein expression in the ovarian tissue of rats with polycystic ovarian syndrome (PCOS) was evaluated. Sixty rats were divided into six groups. Rats in the Dose group received genistein at a concentration of either 5 (L-gen), 10 (M-Gen) or 20 (H-Gen) mg per kg of body weight per day. The expression of Bcl-2 mRNA and Bax mRNA was determined by in situ hybridization. Bcl-2 and Bax protein concentration was quantified by ELISA. The results showed that the expression of Bcl-2 mRNA and Bcl-2 protein was significantly higher in the high genistein Dose group (H-Gen) when compared to the Model group (MG) (P<0.05). Genistein induced higher expression of the Bcl-2 gene at the transcriptional and translational level. Treatment with genistein resulted in an improvement of ovarian function with Bcl-2 expression being enhanced and Bax expression being suppressed. These alterations may be due to the structural and functional modifications that take place in these cells, and could be related to apoptotic changes that occur in rats with PCOS.

BAD overexpression inhibits cell growth and induces apoptosis via mitochondrial-dependent pathway in non-small cell lung cancer.

PubMed

Jiang, Li; Luo, Man; Liu, Dan; Chen, Bojiang; Zhang, Wen; Mai, Lin; Zeng, Jing; Huang, Na; Huang, Yi; Mo, Xianming; Li, Weimin

2013-06-01

The pro-apoptotic Bcl-2 protein BAD initiated apoptosis in human cells and has been identified as a prognostic marker in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the functions of BAD in NSCLC. Overexpression of BAD was performed by transfecting different NSCLC cell lines with wild-type BAD. Cell proliferation, cell cycle, apoptosis, and invasion were characterized in vitro. Tumorigenicity was analyzed in vivo. Western blot was performed to determine the effects of BAD overexpression on the Bcl-2 family proteins and apoptosis-related proteins. Overexpression of BAD significantly inhibited cell proliferation in H1299, H292, and SPC-A1 but not in SK-MES-1 and H460 cell lines in vitro. BAD overexpression also reduced the tumorigenicity of H1299/SPC-A1 cell in vivo. However, no appreciable effects on cell cycle distribution and invasion were observed in all these cell lines. BAD overexpression also induced apoptosis in all cell types, in which process expression of mitochondrial cytochrom c (cyto-c) and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax and caspase 8 expressions did not changed. These findings indicated that a mitochondrial pathway, in which process cyto-c was released from mitochondrial to activate caspase 3, was involved in BAD overexpression-mediated apoptosis. Our data suggested that increased expression of BAD enhance apoptosis and has negative influence on cell proliferation and tumor growth in NSCLC. Bad is a new potential target for tumor interventions.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

PubMed

Renault, Thibaud T; Teijido, Oscar; Missire, Florent; Ganesan, Yogesh Tengarai; Velours, Gisèle; Arokium, Hubert; Beaumatin, Florian; Llanos, Raul; Athané, Axel; Camougrand, Nadine; Priault, Muriel; Antonsson, Bruno; Dejean, Laurent M; Manon, Stéphen

2015-07-01

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure-Based Design of Potent Bcl-2/Bcl-xL Inhibitors with Strong in Vivo Antitumor Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Haibin; Aguilar, Angelo; Chen, Jianfang

Bcl-2 and Bcl-xL are key apoptosis regulators and attractive cancer therapeutic targets. We have designed and optimized a class of small-molecule inhibitors of Bcl-2 and Bcl-xL containing a 4,5-diphenyl-1H-pyrrole-3-carboxylic acid core structure. A 1.4 {angstrom} resolution crystal structure of a lead compound, 12, complexed with Bcl-xL has provided a basis for our optimization. The most potent compounds, 14 and 15, bind to Bcl-2 and Bcl-xL with subnanomolar K{sub i} values and are potent antagonists of Bcl-2 and Bcl-xL in functional assays. Compounds 14 and 15 inhibit cell growth with low nanomolar IC{sub 50} values in multiple small-cell lung cancer cellmore » lines and induce robust apoptosis in cancer cells at concentrations as low as 10 nM. Compound 14 also achieves strong antitumor activity in an animal model of human cancer.« less

Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.

PubMed

He, Congcong; Bassik, Michael C; Moresi, Viviana; Sun, Kai; Wei, Yongjie; Zou, Zhongju; An, Zhenyi; Loh, Joy; Fisher, Jill; Sun, Qihua; Korsmeyer, Stanley; Packer, Milton; May, Herman I; Hill, Joseph A; Virgin, Herbert W; Gilpin, Christopher; Xiao, Guanghua; Bassel-Duby, Rhonda; Scherer, Philipp E; Levine, Beth

2012-01-18

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

Bag-1 and Bcl-2 gene transfer in malignant glioma: modulation of cell cycle regulation and apoptosis.

PubMed

Roth, W; Grimmel, C; Rieger, L; Strik, H; Takayama, S; Krajewski, S; Meyermann, R; Dichgans, J; Reed, J C; Weller, M

2000-04-01

Bag-1 is a heat shock 70 kDa (Hsp70)-binding protein that can collaborate with Bcl-2 in suppressing apoptosis under some conditions. Here, we report that 11 of 12 human glioma cell lines express Bag-1 protein in vitro. Moreover, 15 of 19 human glioblastomas expressed Bag-1 as assessed by immunohistochemistry in primary tumor specimens. To examine the biological effects of Bag-1 in glioma cells, we expressed Bag-1 or Bcl-2 transgenes in 2 human malignant glioma cell lines, LN-18 and LN-229. Bag-1 significantly slowed glioma cell growth and reduced clonogenicity of both cell lines in vitro. Coexpressed Bcl-2 abrogated these effects of Bag-1. Intracranial LN-229 glioma xenografts implanted into nude mice revealed a substantial growth advantage afforded by Bcl-2. Bag-1 had no such effect, either in the absence or presence of Bcl-2. Upon serum starvation in vitro, Bcl-2 prevented cell death whereas Bag-1 did not. Both Bcl-2 and Bag-1 slowed proliferation of serum-starved cells when expressed alone. Importantly, coexpression of Bcl-2 and Bag-1 provided a distinct growth advantage under conditions of serum starvation that is probably the result of (i) the death-preventing activity of Bcl-2 and (ii) the property of Bag-1 to overcome a Bcl-2-mediated enhancement of exit from the cell cycle. In contrast to these Bcl-2/Bag-1 interactions observed under serum starvation conditions, Bag-1 did not further enhance the strong protection from staurosporine-, CD95 (Fas/Apo1) ligand-, Apo2 ligand (TRAIL)- or chemotherapeutic drug-induced apoptosis afforded by Bcl-2. Taken together, these results indicate a role for Bag-1/Bcl-2 interactions in providing a survival advantage to cancer cells in a deprived microenvironment that may be characteristic of ischemic/hypoxic tumors such as human glioblastoma multiforme, and suggest that Bcl-2/Bag-1 interactions also modulate cell proliferation.

Alteration of apoptosis-related genes in postmenopausal women with uterine prolapse.

PubMed

Saatli, Bahadir; Kizildag, Sefa; Cagliyan, Erkan; Dogan, Erbil; Saygili, Ugur

2014-07-01

We aimed to compare expression levels of antiapoptotic and proapoptotic genes in parametrial and vaginal tissues from postmenopausal women with and without pelvic organ prolapse (POP). We hypothesized that the expression of genes that induce apoptosis may be altered in vaginal and parametrial tissues in postmenopausal women with POP. Samples of vaginal and parametrial tissues were obtained from postmenopausal women with (n = 10) and without (n = 10) POP who underwent vaginal or abdominal hysterectomy. Expression levels of antiapoptotic (BCL-2, BCL-XL) and proapoptotic (BAX, BAD) genes were studied by real-time reverse-transcription polymerase chain reaction (RT-PCR). Gene expression levels of BCL-2 (P < 0.001), BCL-XL (P < 0.001), BAX (p = 0.001), and BAD (p = 0.004) were all higher in vaginal tissues from the POP group compared with the non-POP group. Similarly, gene expression levels of BCL-2 (p < 0.001), BCL-XL (p < 0.001), BAX (p < 0.001), and BAD (p < 0.001) in parametrial tissues were also significantly higher in the POP group compared with the non-POP group. Additionally, expression levels of BCL-2 (p = 0.05), BCL-XL (p < 0.05), BAX (p = 0.05), and BAD (p = 0.07) in the POP group were higher in parametrial tissue than in vaginal tissue samples. Antiapoptotic and proapoptotic gene expression levels differed significantly between postmenopausal women with and without POP. Bcl-2 family genes were overexpressed in the parametrium of patients with POP compared with vaginal tissue, suggesting that the processes responsible for POP have a greater effect on parametrial tissue than vaginal tissue during the development of POP.

A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

PubMed Central

Coleman, Carrie B.; McGraw, Jennifer E.; Feldman, Emily R.; Roth, Alexa N.; Keyes, Lisa R.; Grau, Katrina R.; Cochran, Stephanie L.; Waldschmidt, Thomas J.; Liang, Chengyu; Forrest, J. Craig; Tibbetts, Scott A.

2014-01-01

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the

The ASPP interaction network: electrostatic differentiation between pro- and anti-apoptotic proteins.

PubMed

Benyamini, Hadar; Friedler, Assaf

2011-01-01

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C-terminal ankyrin repeats and SH3 domain (ANK-SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl-2, and NFκB. The structure of the complex between ASPP2(ANK-SH3) and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2(ANK-SH3) with Bcl-2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2(ANK-SH3) with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl-2, and NFκB are different, yet lie on the same face of ASPP2(ANK-SH3) . The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein-binding domains. A subset of surface-exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl-2, and NFκB. We also found a gain of positive charge at the non-protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.

Pegasus XL CYGNSS Spacecraft Mate

NASA Image and Video Library

2016-10-28

Inside Building 1555 at Vandenberg Air Force Base in California, the eight NASA Cyclone Global Navigation Satellite System (CYGNSS) spacecraft installed on their deployment module undergo inspections prior to NASA’s Kennedy Space Center in Florida. Processing activities will prepare the spacecraft for launch aboard an Orbital ATK Pegasus XL rocket. When preparations are competed at Vandenberg, the rocket will be transported to Kennedy attached to the Orbital ATK L-1011 carrier aircraft with in its payload fairing. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fin Installation

NASA Image and Video Library

2016-09-21

Technicians prepare to install one of the fins on the Orbital ATK Pegasus XL rocket inside Building 1555 at Vandenberg Air Force Base in California. The fins will provide aerodynamic stability during flight. The rocket is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida, attached to the Orbital ATK L-1011 carrier aircraft with NASA’s Cyclone Global Navigation Satellite System (CYGNSS) in its payload fairing. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fin Installation

NASA Image and Video Library

2016-09-21

Technicians prepare one of the fins for installation on the Orbital ATK Pegasus XL rocket inside Building 1555 at Vandenberg Air Force Base in California. The fins will provide aerodynamic stability during flight. The rocket is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida, attached to the Orbital ATK L-1011 carrier aircraft with NASA’s Cyclone Global Navigation Satellite System (CYGNSS) in its payload fairing. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fin Installation

NASA Image and Video Library

2016-09-21

Technicians prepare to install one of the fins on the Orbital ATK Pegasus XL rocket inside Building 1555 at Vandenberg Air Force Base in California. The fins will provide aerodynamic stability during flight. The rocket is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida attached to the Orbital ATK L-1011 carrier aircraft with NASA’s Cyclone Global Navigation Satellite System (CYGNSS) in its payload fairing. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fairing Arrival

NASA Image and Video Library

2016-10-11

Technicians with Orbital ATK remove the first half of the Pegasus payload fairing for NASA’s Cyclone Global Navigation Satellite System (CYGNSS) from its shipping container and prepare it for the move to nearby Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida aboard the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Activation of the proapoptotic Bcl-2 protein Bax by a small molecule induces tumor cell apoptosis.

PubMed

Zhao, Guoping; Zhu, Yanglong; Eno, Colins O; Liu, Yanlong; Deleeuw, Lynn; Burlison, Joseph A; Chaires, Jonathan B; Trent, John O; Li, Chi

2014-04-01

The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.

Pegasus XL CYGNSS Arrival at CCAFS

NASA Image and Video Library

2016-12-02

The Orbital ATK L-1011 Stargazer aircraft has arrived at the Skid Strip at Cape Canaveral Air Force Station in Florida. Attached beneath the Stargazer is the Orbital ATK Pegasus XL with NASA's Cyclone Global Navigation Satellite System (CYGNSS) on board. CYGNSS was processed and prepared for its mission at Vandenberg Air Force Base in California. CYGNSS is scheduled for its airborne launch aboard the Pegasus XL rocket from the Skid Strip on Dec. 12. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Arrival at CCAFS

NASA Image and Video Library

2016-12-02

The Orbital ATK L-1011 Stargazer aircraft begins its descent to the Skid Strip at Cape Canaveral Air Force Station in Florida. Attached beneath the Stargazer is the Orbital ATK Pegasus XL with NASA's Cyclone Global Navigation Satellite System (CYGNSS) on board. CYGNSS was processed and prepared for its mission at Vandenberg Air Force Base in California. CYGNSS is scheduled for its airborne launch aboard the Pegasus XL rocket from the Skid Strip on Dec. 12. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets

PubMed Central

Weyrich, Andrew S.; Denis, Melvin M.; Schwertz, Hansjorg; Tolley, Neal D.; Foulks, Jason; Spencer, Eliott; Kraiss, Larry W.; Albertine, Kurt H.; McIntyre, Thomas M.

2007-01-01

New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34+ stem cell–derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamycin. In contrast, overexpression of Bcl-3 in a surrogate cell line enhanced clot retraction. These studies identify new features of post-transcriptional gene regulation and signal-dependant protein synthesis in activated platelets that may contribute to thrombus and wound remodeling and suggest that posttranscriptional pathways are targets for molecular intervention in thrombotic disorders. PMID:17110454

MicroRNA-181c targets Bcl-2 and regulates mitochondrial morphology in myocardial cells

PubMed Central

Wang, Hongjiang; Li, Jing; Chi, Hongjie; Zhang, Fan; Zhu, Xiaoming; Cai, Jun; Yang, Xinchun

2015-01-01

Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl-2 family which includes pro- and anti-apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA-181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR-181c could target the 3′ untranslated region of Bcl-2, one of the anti-apoptotic members of the Bcl-2 family. Thus, we have suggested that miR-181c was involved in regulation of Bcl-2. In this study, we investigated this hypothesis using the Dual-Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR-181c. We found that the level of miR-181c was inversely correlated with the Bcl-2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR-181c resulted in significant changes in the levels of caspases, Bcl-2 and cytochrome C in these cells. The increased level of Bcl-2 caused by the decrease in miR-181c protected mitochondrial morphology from the tumour necrosis factor alpha-induced apoptosis. PMID:25898913

Bcl-2 protects tubular epithelial cells from ischemia reperfusion injury by inhibiting apoptosis.

PubMed

Suzuki, Chigure; Isaka, Yoshitaka; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Takabatake, Yoshitsugu; Ito, Takahito; Takahara, Shiro; Imai, Enyu

2008-01-01

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of graft dysfunction in kidney transplantation subjected to ischemia. The mechanism that triggers inflammation and renal injury after ischemia remains to be elucidated; however, cellular stress may induce apoptosis during the first hours and days after transplantation, which might play a crucial role in early graft dysfunction. Bcl-2 is known to inhibit apoptosis induced by the etiological factors promoting ischemia and reperfusion injury. Accordingly, we hypothesized that an augmentation of the antiapoptotic factor Bcl-2 may thus protect tubular epithelial cells by inhibiting apoptosis, thereby ameliorating the subsequent tubulointerstitial injury. We examined the effects of Bcl-2 overexpression on ischemia-reperfusion (I/R) injury using Bcl-2 transgenic mice (Bcl-2 TG) and their wild-type littermates (WT). To investigate the effects of I/R injury, the left renal artery and vein were clamped for 45 min, followed by reperfusion for 0-96 h. Bcl-2 TG exhibited decreased active caspase protein in the tubular cells, which led to a reduction in TUNEL-positive apoptotic cells. Consequently, interstitial fibrosis and phenotypic changes were ameliorated in Bcl-2 TG. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R, and subsequent interstitial injury by inhibiting tubular apoptosis.

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

PubMed Central

Schoneich, Christian; Dremina, Elena; Galeva, Nadezhda; Sharov, Victor

2014-01-01

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cell but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad. PMID:24129924

BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor

PubMed Central

Kyrylkova, Kateryna; Kyryachenko, Sergiy; Biehs, Brian; Klein, Ophir; Kioussi, Chrissa; Leid, Mark

2012-01-01

Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b−/− and Sprouty mutant mice. PMID:22629441

HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells

PubMed Central

Chen, Tian-Rui; Wei, Hai-feng; Song, Dian-Wen; Liu, Tie-Long; Yang, Xing-Hai; Fu, Chuan-Gang; Hu, Zhi-qian; Zhou, Wang; Yan, Wang-Jun; Xiao, Jian-Ru

2017-01-01

B-cell CLL/lymphoma 9 protein (BCL-9), a multi-functional co-factor in Wnt signaling, induced carcinogenesis as well as promoting tumor progression, metastasis and chemo-resistance in colorectal cancer (CRC). However, the mechanisms for increased BCL-9 expression in CRC were not well understood. Here, we report that hypoxia, a hallmark of solid tumors, induced BCL-9 mRNA expression in human CRC cells. Analysis of BCL-9 promoter revealed two functional hypoxia-responsive elements (HRE-B and HRE-C) that can be specifically bound with and be transactivated by hypoxia inducible factors (HIF) -1α but not HIF-2α. Consistently, ectopic expression of HIF-1α but not HIF-2α transcriptionally induced BCL-9 expression levels in cells. Knockdown of endogenous HIF-1α but not HIF-2α by siRNA largely abolished the induction of HIF by hypoxia. Furthermore, there was a strong association of HIF-1α expression with BCL-9 expression in human CRC specimens. In summary, results from this study demonstrated that hypoxia induced BCL-9 expression in human CRC cells mainly through HIF-1α, which could be an important underlying mechanism for increased BCL-9 expression in CRC. PMID:27121066

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

PubMed

Ji, Haofeng; Shen, Xiuda; Gao, Feng; Ke, Bibo; Freitas, Maria Cecilia S; Uchida, Yoichiro; Busuttil, Ronald W; Zhai, Yuan; Kupiec-Weglinski, Jerzy W

2010-10-01

Programmed death-1 (PD-1)/B7-H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity-dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD-1/B7-H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD-1 signaling after anti-B7-H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7-H1 immunoglobulin (B7-H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well-preserved liver architecture. The therapeutic potential of B7-H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti-necrotic/apoptotic Bcl-2/Bcl-xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)-10. Neutralization of IL-10 re-created liver IRI and rendered B7-H1Ig-treated hosts susceptible to IRI. These findings were confirmed in T cell-macrophage in vitro coculture in which B7-H1Ig diminished tumor necrosis factor-α/IL-6 levels in an IL-10-dependent manner. Our novel findings document the essential role of the PD-1/B7-H1 pathway in liver IRI. This study is the first to demonstrate that stimulating PD-1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD-1 upon T cell-Kupffer cell cross-talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL-10-dependent cytoprotection.

The Role of the Acidity of N-Heteroaryl Sulfonamides as Inhibitors of Bcl-2 Family Protein–Protein Interactions

PubMed Central

2013-01-01

Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263. PMID:24900652

The transcription factor Wilms tumor 1 confers resistance in myeloid leukemia cells against the proapoptotic therapeutic agent TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand) by regulating the antiapoptotic protein Bcl-xL.

PubMed

Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

2012-09-21

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

PubMed Central

Colin, Didier J.; Hain, Karolina O.; Allan, Lindsey A.; Clarke, Paul R.

2015-01-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies. PMID:25761368

Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

PubMed

Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

2007-08-01

Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

Pegasus XL CYGNSS Fairing Mate and Black Light Test

NASA Image and Video Library

2016-11-14

Technicians with Orbital ATK perform a black light test on the Pegasus XL fairing inside Building 1555 at Vandenberg Air Force Base in California. NASA’s Cyclone Global Navigation Satellite System (CYGNSS) is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida aboard the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 Stargazer aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Fairing Mate and Black Light Test

NASA Image and Video Library

2016-11-14

Technicians with Orbital ATK install the first half of the Pegasus XL fairing around NASA’s Cyclone Global Navigation Satellite System (CYGNSS) in Building 1555 at Vandenberg Air Force Base in California. CYGNSS is being prepared at Vandenberg, and then will be transported to NASA’s Kennedy Space Center in Florida aboard the Orbital ATK Pegasus XL rocket which will be attached to the Orbital ATK L-1011 carrier aircraft. CYGNSS will launch on the Pegasus XL rocket from the Skid Strip at Cape Canaveral Air Force Station. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Pegasus XL CYGNSS Arrival at CCAFS

NASA Image and Video Library

2016-12-02

The Orbital ATK L-1011 Stargazer aircraft touches down at 3:57 p.m. EST at the Skid Strip at Cape Canaveral Air Force Station in Florida. Attached beneath the Stargazer is the Orbital ATK Pegasus XL with NASA's Cyclone Global Navigation Satellite System (CYGNSS) on board. CYGNSS was processed and prepared for its mission at Vandenberg Air Force Base in California. CYGNSS is scheduled for its airborne launch aboard the Pegasus XL rocket from the Skid Strip on Dec. 12. CYGNSS will make frequent and accurate measurements of ocean surface winds throughout the life cycle of tropical storms and hurricanes. The data that CYGNSS provides will enable scientists to probe key air-sea interaction processes that take place near the core of storms, which are rapidly changing and play a critical role in the beginning and intensification of hurricanes.

Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages

PubMed Central

Zhao, Xiaomin; Khan, Nargis; Gan, Huixian; Tzelepis, Fanny; Nishimura, Tomoyasu; Park, Seung-Yeol; Divangahi, Maziar; Remold, Heinz G.

2017-01-01

Virulent Mycobacterium tuberculosis (Mtb) triggers necrosis in host Mφ, which is essential for successful pathogenesis. Here we demonstrate that necrosis of Mtb-infected Mφ is dependent on the action of the cytosolic kinase Receptor Interacting Protein 3 (RIPK3) and the mitochondrial Bcl-2 family member protein B-cell lymphoma - extra large (Bcl-xL). RIPK3-deficient Mφ are able to better control bacterial growth in vitro and in vivo. Cytosolic RIPK3 translocates to the mitochondria where it promotes necrosis and blocks caspase 8-activation and apoptosis via Bcl-xL. Furthermore, necrosis is associated with stabilization of hexokinase II on the mitochondria as well as cyclophilin D-dependent mitochondrial permeability transition (MPT). These events up-regulate the level of reactive oxygen species (ROS) to induce necrosis. Thus, in Mtb-infected Mφ mitochondria are an essential platform for induction of necrosis by activating RIPK3 function and preventing caspase 8 - activation. PMID:28401933

Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages.

PubMed

Zhao, X; Khan, N; Gan, H; Tzelepis, F; Nishimura, T; Park, S-Y; Divangahi, M; Remold, H G

2017-11-01

Virulent Mycobacterium tuberculosis (Mtb) triggers necrosis in host Mϕ, which is essential for successful pathogenesis in tuberculosis. Here we demonstrate that necrosis of Mtb-infected Mϕ is dependent on the action of the cytosolic Receptor Interacting Protein Kinase 3 (RIPK3) and the mitochondrial Bcl-2 family member protein B-cell lymphoma-extra large (Bcl-x L ). RIPK3-deficient Mϕ are able to better control bacterial growth in vitro and in vivo. Mechanistically, cytosolic RIPK3 translocates to the mitochondria where it promotes necrosis and blocks caspase 8-activation and apoptosis via Bcl-x L . Furthermore, necrosis is associated with stabilization of hexokinase II on the mitochondria as well as cyclophilin D-dependent mitochondrial permeability transition. Collectively, these events upregulate the level of reactive oxygen species to induce necrosis. Thus, in Mtb-infected Mϕ, mitochondria are an essential platform for induction of necrosis by activating RIPK3 function and preventing caspase 8-activation.

Adenoviral bcl-2 transfer improves survival and early graft function after ischemia and reperfusion in rat liver transplantation.

PubMed

Rentsch, Markus; Kienle, Klaus; Mueller, Thomas; Vogel, Mandy; Jauch, Karl Walter; Püllmann, Kerstin; Obed, Aiman; Schlitt, Hans J; Beham, Alexander

2005-11-27

Primary graft dysfunction due to ischemia and reperfusion injury represents a major problem in liver transplantation. The related cell stress may induce apoptosis, which can be suppressed by bcl-2. The purpose of the study was to investigate the effect of adenoviral bcl-2 gene transfer on early graft function and survival in rat liver transplantation. An adenoviral construct that transfers bcl-2 under the control of a tetracycline inducible promoter was generated (advTetOn bcl-2) and used with a second adenovirus that transfers the repressor protein (advCMV Rep). Forty-eight hours before explantation, donor rats were treated with advTetOn bcl-2/ advCMV Rep (n=7) and doxycyclin, with the control adenoviral construct advCMV GFP (n=8) or with doxycyclin alone (n=8). Liver transplantation was performed following 16 hours of cold storage (UW). Bcl-2 expression and intrahepatic apoptosis was assessed. Bile flow was monitored 90 min posttransplantation. The endpoint for survival was 7 days. Bcl-2 was expressed in hepatocytes and sinusoidal lining cells. This was associated with a significant reduction of apoptotic sinusoidal lining cells and hepatocytes after 24 hours and 7 days. Bile production was significantly higher following bcl-2 pretreatment. Furthermore, bcl-2 transfer resulted in significantly improved survival (100% vs. 50% both control groups). Adenoviral bcl-2 transfer results in protein expression in hepatocytes and sinusoidal lining cells resulting in early graft function and survival enhancement after prolonged ischemia and reperfusion injury. The inhibition of apoptosis in the context of liver transplantation might be a reasonable approach in the treatment of graft dysfunction.

Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

PubMed Central

Zhao, Guoping; Zhu, Yanglong; Eno, Colins O.; Liu, Yanlong; DeLeeuw, Lynn; Burlison, Joseph A.; Chaires, Jonathan B.; Trent, John O.

2014-01-01

The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer. PMID:24421393

Akt-dependent glucose metabolism promotes Mcl-1 synthesis to maintain cell survival and resistance to Bcl-2 inhibition.

PubMed