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

Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma.

PubMed

Tamura, A; Miura, I; Iida, S; Yokota, S; Horiike, S; Nishida, K; Fujii, H; Nakamura, S; Seto, M; Ueda, R; Taniwaki, M

2001-08-01

To detect immunoglobulin heavy chain (IGH) gene translocations with specific oncogene loci, we established an interphase cytogenetic approach using double-color fluorescence in situ hybridization (DC-FISH), which we used to analyze 173 patients with B-cell lymphoma. DC-FISH using the IGH gene (14q32.3) in combination with c-MYC (8q24.1), BCL1 (11q13.3), BCL2 (18q21.3), BCL6 (3q27), and PAX-5 (9p13) gene probes detected IGH translocations in 70 (40.5%) of 173 patients. The partner genes involved in IGH translocations were identified in 56 (80%) of 70 patients, and fusion of the IGH gene with specific oncogenes was detected in 53 of 56 patients, particularly in interphase nuclei of 28 patients for whom cytogenetic analysis was not informative. The most common partner gene was BCL2 (19 patients; 27% of IGH translocation-positive patients), followed by BCL6 (16; 23%), BCL1 (11; 16%), c-MYC (7; 10%), and PAX-5 (2; 3%). These oncogenes were closely associated with subtypes of B-cell lymphoma. The other partners were 19q13 (BCL3), 6p25 (MUM1/IRF4), 1q36, and chromosome 8 identified in one patient each. Six of the nine patients with add(14)(q32) showed a BCL6/IGH translocation. Double translocations of the IGH gene were found in three patients; c-MYC+BCL1, c-MYC+BCL2, and c-MYC+BCL6 in each one. Interphase FISH using specific IGH-translocation probes is valuable for defining clinically meaningful subgroups of B-cell lymphoma.

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

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.

Efficacy of venetoclax as targeted therapy for relapsed/refractory t(11;14) multiple myeloma.

PubMed

Kumar, Shaji; Kaufman, Jonathan L; Gasparetto, Cristina; Mikhael, Joseph; Vij, Ravi; Pegourie, Brigitte; Benboubker, Lofti; Facon, Thierry; Amiot, Martine; Moreau, Philippe; Punnoose, Elizabeth A; Alzate, Stefanie; Dunbar, Martin; Xu, Tu; Agarwal, Suresh K; Enschede, Sari Heitner; Leverson, Joel D; Ross, Jeremy A; Maciag, Paulo C; Verdugo, Maria; Touzeau, Cyrille

2017-11-30

Venetoclax is a selective, orally bioavailable BCL-2 inhibitor that induces cell death in multiple myeloma (MM) cells, particularly in those harboring t(11;14), which express high levels of BCL-2 relative to BCL-X L and MCL-1. In this phase 1 study, patients with relapsed/refractory MM received venetoclax monotherapy. After a 2-week lead-in with weekly dose escalation, daily venetoclax was given at 300, 600, 900, or 1200 mg in dose-escalation cohorts and 1200 mg in the safety expansion. Dexamethasone could be added on progression during treatment. Sixty-six patients were enrolled (30, dose-escalation cohorts; 36, safety expansion). Patients received a median of 5 prior therapies (range, 1-15); 61% were bortezomib and lenalidomide double refractory, and 46% had t(11;14). Venetoclax was generally well tolerated. Most common adverse events included mild gastrointestinal symptoms (nausea [47%], diarrhea [36%], vomiting [21%]). Cytopenias were the most common grade 3/4 events, with thrombocytopenia (32%), neutropenia (27%), anemia (23%), and leukopenia (23%) reported. The overall response rate (ORR) was 21% (14/66), and 15% achieved very good partial response or better (≥VGPR). Most responses (12/14 [86%]) were reported in patients with t(11;14). In this group, ORR was 40%, with 27% of patients achieving ≥VGPR. Biomarker analysis confirmed that response to venetoclax correlated with higher BCL2:BCL2L1 and BCL2:MCL1 mRNA expression ratios. Venetoclax monotherapy at a daily dose up to 1200 mg has an acceptable safety profile and evidence of single-agent antimyeloma activity in patients with relapsed/refractory MM, predominantly in patients with t(11;14) abnormality and those with a favorable BCL2 family profile. Registered at www.clinicaltrials.gov: #NCT01794520. © 2017 by The American Society of Hematology.

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

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.

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.

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

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

PubMed Central

Estruch, Sara B.; Buzón, Víctor; Carbó, Laia R.; Schorova, Lenka; Lüders, Jens; Estébanez-Perpiñá, Eva

2012-01-01

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain. PMID:22675500

The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

PubMed

Estruch, Sara B; Buzón, Víctor; Carbó, Laia R; Schorova, Lenka; Lüders, Jens; Estébanez-Perpiñá, Eva

2012-01-01

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Dendritic cell fate is determined by BCL11A

PubMed Central

Ippolito, Gregory C.; Dekker, Joseph D.; Wang, Yui-Hsi; Lee, Bum-Kyu; Shaffer, Arthur L.; Lin, Jian; Wall, Jason K.; Lee, Baeck-Seung; Staudt, Louis M.; Liu, Yong-Jun; Iyer, Vishwanath R.; Tucker, Haley O.

2014-01-01

The plasmacytoid dendritic cell (pDC) is vital to the coordinated action of innate and adaptive immunity. pDC development has not been unequivocally traced, nor has its transcriptional regulatory network been fully clarified. Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and demonstrate this lineage-specific requirement in the adult organism. Furthermore, we identify BCL11A gene targets and provide a molecular mechanism for its action in pDC commitment. Embryonic germ-line deletion of Bcl11a revealed an absolute cellular, molecular, and functional absence of pDCs in fetal mice. In adults, deletion of Bcl11a in hematopoietic stem cells resulted in perturbed yet continued generation of progenitors, loss of downstream pDC and B-cell lineages, and persisting myeloid, conventional dendritic, and T-cell lineages. Challenge with virus resulted in a marked reduction of antiviral response in conditionally deleted adults. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators, including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development, supporting a model wherein differentiation into pDCs represents a primed “default” pathway for common dendritic cell progenitors. PMID:24591644

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.

Neoplastic MiR-17~92 deregulation at a DNA fragility motif (SIDD).

PubMed

Schneider, Björn; Nagel, Stefan; Ehrentraut, Stefan; Kaufmann, Maren; Meyer, Corinna; Geffers, Robert; Drexler, Hans G; MacLeod, Roderick A F

2012-03-01

Chromosomal or mutational activation of BCL6 (at 3q27) typifies diffuse large B-cell lymphoma (DLBCL) which in the germinal center subtype may be accompanied by focal amplification of chromosome band 13q31 effecting upregulation of miR-17~92. Using long distance inverse-polymerase chain reaction, we mapped and sequenced six breakpoints of a complex BCL6 rearrangement t(3;13)(q27;q31)t(12;13)(p11;q31) in DLBCL cells, which places miR-17~92 antisense within the resulting ITPR2-BCL6 chimeric fusion gene rearrangement. MiR-17~92 members were upregulated ~15-fold over controls in a copy number independent manner consistent with structural deregulation. MIR17HG and ITPR2-BCL6 were, despite their close configuration, independently expressed, discounting antisense regulation. MIR17HG in t(3;13)t(12;13) cells proved highly responsive to treatment with histone deacetylase inhibitors implicating epigenetic deregulation, consistent with which increased histone-H3 acetylation was detected by chromatin immunoprecipitation near the upstream MIR17HG breakpoint. Remarkably, 5/6 DNA breaks in the t(3;13)t(12;13) precisely cut at stress-induced DNA duplex destabilization (SIDD) peaks reminiscent of chromosomal fragile sites, while the sixth lay 150 bp distant. Extended SIDD profiling showed that additional oncomiRs also map to SIDD peaks. Fluorescence in situ hybridization analysis showed that 11 of 52 (21%) leukemia-lymphoma (L-L) cell lines with 13q31 involvement bore structural rearrangements at/near MIR17HG associated with upregulation. As well as fueling genome instability, SIDD peaks mark regulatory nuclear-scaffold matrix attachment regions open to nucleosomal acetylation. Collectively, our data indict a specific DNA instability motif (SIDD) in chromosome rearrangement, specifically alterations activating miR-17~92 epigenetically via promoter hyperacetylation, and supply a model for the clustering of oncomiRs near cancer breakpoints. Copyright © 2011 Wiley-Liss, Inc.

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.

Long Noncoding RNA HOTAIR Modulates MiR-206-mediated Bcl-w Signaling to Facilitate Cell Proliferation in Breast Cancer.

PubMed

Ding, Wei; Ren, Jin; Ren, Hui; Wang, Dan

2017-12-08

LncRNA HOX transcript antisense RNA (HOTAIR) is involved in lots of cancers. The pro-survival protein Bcl-w is frequently found in cancer development. However, the effect of HOTAIR on Bcl-w in breast cancer is not well documented. In this study, we first evaluated the correlation between HOTAIR level and Bcl-w expression in clinical breast cancer tissues. We observed that the expression levels of Bcl-w were much higher in the breast cancer samples than that in their paired noncancerous tissues. Moreover, the levels of HOTAIR were positively associated with those of Bcl-w in clinical breast cancer samples. As expected, we observed that HOTAIR was able to up-regulate the expression of Bcl-w in breast cancer cells. Mechanistically, we found that miR-206 was capable of inhibiting the expression of Bcl-w by directly binding to the 3'UTR of Bcl-w mRNA. Interestingly, HOTAIR could increase the expression of Bcl-w through sequestering miR-206 at post-transcriptional level. Functionally, our data showed that HOTAIR-induced Bcl-w by miR-206 facilitated the proliferation of breast cancer cells. Thus, we conclude that HOTAIR up-regulates Bcl-w to enhance cell proliferation through sequestering miR-206 in breast cancer. Our finding provides new insights into the mechanism of breast cancer mediated by HOTAIR.

MicroRNA-9 Mediates the Cell Apoptosis by Targeting Bcl2l11 in Ischemic Stroke.

PubMed

Wei, Na; Xiao, Lin; Xue, Rui; Zhang, Dandan; Zhou, Jun; Ren, Huayan; Guo, Si; Xu, Jingjing

2016-12-01

Ischemic strokes occur as a result of an obstruction within a blood vessel supplying blood to the brain and accounts for about 87 % of all cases. During the cerebral ischemia, most of the neurons undergo the necrosis and apoptosis upon the exposure to the dramatic blood flow reduction. Although, it is known that both the intrinsic and extrinsic pathways are involved in the neuronal apoptosis of ischemic brain injury. The complex underlying mechanisms remains less known. MicroRNAs are a class of endogenous small non-coding RNAs and the role of miRNAs in the pathophysiology of stroke has been studied. In this study, we found that miR-9 is downregulated in the mice with middle cerebral artery occlusion (MCAO) brain and oxygen-glucose deprivation (OGD) neurons. Application of miR-9 gamer could restore the neurological scores and reduces the infarct volume, brain water content, and the behavioral impairments. Moreover, upregulation of miR-9 suppresses the neuronal apoptosis in MCAO brain and OGD neurons. Furthermore, we identified that Bcl2l11 as the direct target of miR-9 and manipulation of miR-9 induces the corresponding changing of Bcl2l11 protein level. Finally, we found that the protein level of Bcl2l11 is increased in the MCAO brain and OGD neurons. Our study demonstrated the critical role of miR-9 in the neuronal apoptosis of ischemic brain injury.

Clinicopathological and genomic analysis of double-hit follicular lymphoma: comparison with high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements.

PubMed

Miyaoka, Masashi; Kikuti, Yara Y; Carreras, Joaquim; Ikoma, Haruka; Hiraiwa, Shinichiro; Ichiki, Akifumi; Kojima, Minoru; Ando, Kiyoshi; Yokose, Tomoyuki; Sakai, Rika; Hoshikawa, Masahiro; Tomita, Naoto; Miura, Ikuo; Takata, Katsuyoshi; Yoshino, Tadashi; Takizawa, Jun; Bea, Silvia; Campo, Elias; Nakamura, Naoya

2018-02-01

Most high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements are aggressive B-cell lymphomas. Occasional double-hit follicular lymphomas have been described but the clinicopathological features of these tumors are not well known. To clarify the characteristics of double-hit follicular lymphomas, we analyzed 10 cases of double-hit follicular lymphomas and 15 cases of high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements for clinicopathological and genome-wide copy-number alterations and copy-neutral loss-of-heterozygosity profiles. For double-hit follicular lymphomas, the median age was 67.5 years (range: 48-82 years). The female/male ratio was 2.3. Eight patients presented with advanced clinical stage. The median follow-up time was 20 months (range: 1-132 months). At the end of the follow-up, 8 patients were alive, 2 patients were dead including 1 patient with diffuse large B-cell lymphoma transformation. Rearrangements of MYC/BCL2, MYC/BCL6, and MYC/BCL2/BCL6 were seen in 8, 1, and 1 cases, respectively. The partner of MYC was IGH in 6 cases. There were no cases of histological grade 1, 4 cases of grade 2, 5 cases of grade 3a, and 1 case of grade 3b. Two cases of grade 3a exhibited immunoblast-like morphology. Immunohistochemistry demonstrated 9 cases with ≥50% MYC-positive cells. There was significant difference in MYC intensity (P=0.00004) and MIB-1 positivity (P=0.001) between double-hit follicular lymphomas and high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements. The genome profile of double-hit follicular lymphomas was comparable with conventional follicular lymphomas (GSE67385, n=198) with characteristic gains of 2p25.3-p11.1, 7p22.3-q36.3, 12q11-q24.33, and loss of 18q21.32-q23 (P<0.05). In comparison with high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements, double-hit follicular lymphomas had fewer copy-number alterations and minimal common region of gain at 2p16.1 (70%), locus also significant against conventional follicular lymphomas (P=0.0001). In summary, double-hit follicular lymphomas tended to be high-grade histology, high MYC protein expression, high MYC/IGH fusion, and minimal common region of gain at 2p16.1. Double-hit follicular lymphomas seemed to be a different disease from high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements and have an indolent clinical behavior similar to follicular lymphomas without MYC rearrangement.

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

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

Development of large diameter carbon monofilament

NASA Technical Reports Server (NTRS)

Jacob, B.; Neltri, R. D.

1973-01-01

A process for preparing large diameter carbon-boron monofilament was developed. The process involves chemical vapor depositing a carbon-boron alloy monofilament from a BCl3, CH4, and H2 gas mixture onto a carbon substrate. Amorphous alloys were formed when gaseous mixtures containing greater than 20 percent methane (80 percent BCl3) were used. The longest uninterrupted lengths of carbon-boron monofilament were produced using a CH4/BCl3 gas ratio of 2.34. It was found that the properties of the carbon-boron alloy monofilament improved when the carbon substrate was precleaned in chlorine. The highest strength monofilament was attained when a CH4/BCl3 gas volume ratio of 0.44 was 28 million N/sq cm (40 million psi). While the highest strengths were attained in this run, the 0.44 gas ratio and other CH4/BCl3 ratios less than 2.34 would not yield long runs. Runs using these ratios were usually terminated because of a break in the monofilament within the reactor. It is felt better process control could probably be achieved by varying the amount of hydrogen; the BCl3/H2 ratio was kept constant in these studies.

Molecular cloning of IGλ rearrangements using long-distance inverse PCR (LDI-PCR).

PubMed

Shimanuki, Masaya; Sonoki, Takashi; Hosoi, Hiroki; Watanuki, Jyuri; Murata, Shogo; Kawakami, Keiki; Matsuoka, Hiroshi; Hanaoka, Nobuyoshi; Nakakuma, Hideki

2013-01-01

Malignant cells of mature B-cell origin show tumor-specific clonal immunoglobulin gene (IG) rearrangements, including V(D)J recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long-distance inverse PCR (LDI-PCR) has so far been applied to rearrangements targeted to IGH joining, IGH switch, and IGκ regions. We tended to apply LDI-PCR method for cloning of IGλ rearrangements. To identify which IGλ isotype segment was rearranged, we performed Southern blot analysis using isotype-specific probes. We set inverse primers on the telomeric side of each joining region and amplified rearranged bands detected by Southern blot analysis as corresponding PCR products. All germline IGλ segments were successfully amplified as expected PCR products. We determined breakpoint sequences of five chromosome translocations involving IGλ locus: three novel t(8;22)(q24;q11), one known t(3;22)(q27;q11), and one partially known t(11;22)(q13;q11). Two of the three t(8;22)(q24;q11) were involved in Jλ with a recombination signal sequence and one of three in the first exon of IGLL5, which lies upstream of Jλ1. Three 8q24 breakpoints were widespread at 132, 260 and 366 kb downstream of MYC locus. The t(3;22)(q27;q11) showed a juxtaposition of Jλ2 and the first intron of BCL6, as previously reported. In t(11;22)(q13;q11), 3'UTR of cyclin D1 fused to the constant region of λ7 with nucleotide mutations. We also amplified four Vλ/Jλ recombination sequences. Our method is a useful tool for molecular analysis of genetic events in IGλ. © 2012 John Wiley & Sons A/S.

Fluorescence immunophenotyping and interphase cytogenetics (FICTION) detects BCL6 abnormalities, including gene amplification, in most cases of nodular lymphocyte-predominant Hodgkin lymphoma.

PubMed

Bakhirev, Alexei G; Vasef, Mohammad A; Zhang, Qian-Yun; Reichard, Kaaren K; Czuchlewski, David R

2014-04-01

BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique.

Bax and Bid act in synergy to bring about T11TS-mediated glioma apoptosis via the release of mitochondrial cytochrome c and subsequent caspase activation.

PubMed

Bhattacharjee, M; Acharya, S; Ghosh, A; Sarkar, P; Chatterjee, S; Kumar, P; Chaudhuri, S

2008-12-01

The specific apoptotic role of T11TS has been well established in glioma animal models. T11TS specifically induces the glioma cells to die an apoptotic death via immune cross-talk with the two intracranial immune competent cells-microglia and the brain-infiltrating lymphocytes. To unearth the molecular cascades operative within the glioma cells and to some extent in the two interacting immunocytes, we had initiated studies where preliminary findings not only had indicated the involvement of death receptors but had also hinted to the involvement of other apoptotic regulators. Hence, to identify the molecular pathway of apoptosis involving other apoptotic regulators in the three cell types, the cells were studied for the intrinsic apoptotic death regulators that were engaged to maintain the mitochondrial membrane integrity. The proteins that were selected could be divided into three broad classes-the Bcl-2 family of proteins-Bid, Bax and Bcl-2; the guardian of the genome p53 and the proteins downstream of mitochondria-Apaf-1, cytochrome c, caspase-9 and caspase-3. Activated Bid as well as maximal p53 expression was observed in the first dose of T11TS thus dually activating the pro-apoptotic Bax in the first and second dose in the glioma cells. Concurrently, the pro-survival protein Bcl-2's expression level was very much down-regulated in the same two doses favoring the internal microenvironment to proceed for apoptosis. High expression of cytochrome c and Apaf-1 and the presence of active caspase-9 and active caspase-3 in all the T11TS-treated tumor-bearing groups further adjudicated apoptosis of the glioma cells with clear involvement of mitochondrial death pathway in the T11TS-treated animals. Even though expression of the apoptotic regulators remained more or less the same indicating the involvement of mitochondria in the two interacting immunocytes, the intensity of expression of these proteins was much lower than the tumor cells. The present work focuses on the mechanistic approach of how T11TS mediates apoptosis and hence is the first approach of its kind in the field of immunology where the immunotherapeutic molecule's mode of action has been worked out.

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

PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma.

PubMed Central

Rosenberg, C L; Wong, E; Petty, E M; Bale, A E; Tsujimoto, Y; Harris, N L; Arnold, A

1991-01-01

Rearrangement of the BCL1 (B-cell lymphoma 1) region on chromosome 11q13 appears to be highly characteristic of centrocytic lymphoma and also is found infrequently in other B-cell neoplasms. Rearrangement is thought to deregulate a nearby protooncogene, but transcribed sequences in the immediate vicinity of BCL1 breakpoints had not been identified. PRAD1, previously designated D11S287E, was identified on 11q13 as a chromosomal breakpoint region rearranged with the parathyroid hormone gene in a subset of parathyroid adenomas; this highly conserved putative oncogene, which encodes a novel cyclin, has been linked to BCL1 and implicated also in subsets of breast and squamous cell neoplasms with 11q13 amplification. We report pulsed-field gel electrophoresis data showing BCL1 and PRAD1 to be no more than 130 kilobases apart. PRAD1 mRNA is abundantly expressed in seven of seven centrocytic lymphomas (Kiel classification), in contrast to 13 closely related but noncentrocytic lymphomas. Three of the seven centrocytic lymphomas had detectable BCL1 DNA rearrangement. Also, two unusual cases of CLL with BCL1 rearrangement overexpressed PRAD1, in contrast to five CLL controls. Thus, PRAD1 is an excellent candidate "BCL1 oncogene." Its overexpression may be a key consequence of rearrangement of the BCL1 vicinity in B-cell neoplasms and a unifying pathogenetic feature in centrocytic lymphoma. Images PMID:1682919

PRAD1, a candidate BCL1 oncogene: Mapping and expression in centrocytic lymphoma

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

Rosenberg, C.L.; Arnold, A.; Harris, N.L.

Rearrangement of the BCL1 (B-cell lymphoma 1) region on chromosome 11q13 appears to be highly characteristic of centrocytic lymphoma and also is found infrequently in other B-cell neoplasma. Rearrangement is thought to deregulate a nearby protooncogene, but transcribed sequences in the immediate vicinity of BCL1 breakpoints had not been identified. PRAD1, previously designated D11S287E, was identified on 11q13 as a chromosomal breakpoint region rearranged with the parathyroid hormone gene in a subset of parathyroid adenomas; this highly conserved putative oncogene, which encodes a novel cyclin, has been linked to BCL1 and implicated also in subsets of breast and squamous cellmore » neoplasms with 11q13 amplification. The authors report pulsed-field gel electrophoresis data showing BCL1 and PRAD1 to be no more than 130 kilobases apart. PRAD1 mRNA is abundantly expressed in seven of seven centrocytic lymphomas (Kiel classification), in contrast to 13 closely related but noncentrocytic lymphomas. Three of the seven centrocytic lymphomas had detectable BCL1 DNA rearrangement. Also, two unusual cases of CLL and BCL1 rearrangement overexpressed PRAD1, in contrast to five CLL controls. Thus, PRAD1 is an excellent candidate BCL1 oncogene. Its overexpression may be a key consequence of rearrangement of the BCL1 vicinity in B-cell neoplasms and a unifying pathogenetic feature in centrocytic lymphoma.« less

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

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 mimetics warrants attention in AML, particularly in the setting of basal AKT activation and/or addiction.

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.

[Primary central nervous system diffuse large B cell lymphoma: a clinicopathologic and molecular study].

PubMed

Ma, Z P; Ainiwaer, Babayi; Liu, Z Y; Shi, X L; Cui, W L; Zhang, W; Li, X X

2016-11-08

Objective: To investigate clinicopathologic characteristics, immunophenotype and EB virus-related molecular genetic alterations in primary central nervous system diffuse large B cell lymphoma (DLBCL) along with correlation with clinical prognosis. Methods: A total of 30 cases of primary central nervous system DLBCL were retrospectively studied by retrieving clinical data, histological evaluation and immunophenotyping by EnVision two steps methods. The expression of EBER mRNA was detected by in situ hybridization and bcl-2, bcl-6 and C-MYC gene abnormalities were analyzed by interphase fluorescence in situ hybridization. Results: The cases included 18 males and 12 females (sex ratio of 1.5∶1.0) with an age ranging from 24 to 78 years (average age of 52 years, the median age of 53 years). The single primary clinical presentation was focal neurologic deficits. Tumor locations were supratentorial (21 cases), subtentorial (7 cases), involving both locations in 2 cases. Diffuse growth pattern was observed with large lymphoid cells mostly resembling centroblasts with abundant basophilic cytoplasm with oval to round, vesicular nuclei containing fine chromatin. An angiocentric and angiodestructive growth pattern was also present. Other features included perivascular space invasion. Immunohistochemical staining using a panel of CD10, bcl-6 and MUM1, six cases were germinal center-like (GCB) and 24 cases were non-germinal central-like (non-GCB). The positive rates of bcl-2, bcl-6 and C-MYC were 53.3% (16/30), 80.0% (24/30) and 20.0% (6/30), respectively. Genetic alterations were detected by FISH and the gene arrangement rates of bcl-2, bcl-6 and C-MYC were 3.3% (1/30), 16.7% (5/30) and 3.3% (1/30), respectively. There were 19 cases in stage 0-1 disease and 11 cases had stage 2-3 disease. Postoperative follow-up for average 13.6 months showed the median survival of 10 months, one-year survival of 46.7% and 16 patients died within a year. Conclusions: The clinical prognosis of primary central nerve system DLBCL depends on age, clinical performence status score, IPI score, immune classification and treatment. Patients typically progress rapidly with the high mortality within one year of diagnosis. Surgical resection combined with high-dose methotrexate or cytarabine chemotherapy offer the best treatment option.

Artemisia leaf extract induces apoptosis in human endometriotic cells through regulation of the p38 and NFκB pathways.

PubMed

Kim, Ji-Hyun; Jung, Seung-Hyun; Yang, Yeong-In; Ahn, Ji-Hye; Cho, Jin-Gyeong; Lee, Kyung-Tae; Baek, Nam-In; Choi, Jung-Hye

2013-02-13

Artemisia leaves have long been used for the treatment of gynecological disorders, including infertility and dysmenorrhea, which can be commonly caused by endometriosis. In the present study, we investigated the effect of Artemisia princeps extract (APE) on the cell growth and apoptosis of human endometriotic cells. MTT assays and FACS analysis using PI and Annexin staining were performed to study cell viability, cell cycle progression, and apoptosis. We also explored the mechanism of APE-induced effects by evaluating the activation of caspases, Akt, p38, and NFκB. The expressions of XIAP, Bcl-2, and Bcl-xL were measured by real-time RT-PCR and Western blot analyses. APE significantly inhibited the cell viability of 11Z and 12Z human endometriotic epithelial cells. Interestingly, endometriotic cells were more sensitive to APE treatment than immortalized endometrial cells (HES). Treatment with APE induced apoptosis of 11Z cells in a time-dependent manner, as shown by accumulation of sub G1 and apoptotic cell populations. In addition, treatment with APE stimulated the activation of caspase -3, -8, and -9 in a dose- and time-dependent manner. Furthermore, p38 was activated by APE treatment, and the p38 inhibitor SB203580 markedly inhibited APE-induced cell death in 11Z cells. Moreover, treatment with APE suppressed the activation of NFκB and the expressions of anti-apoptotic factors such as XIAP, Bcl-2, and Bcl-xL. These results indicate that APE is a potential anti-endometriotic agent, acting to induce apoptosis of endometrial cells through the modulation of the p38 and NFκB pathways. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Characterization of a candidate bcl-1 gene.

PubMed Central

Withers, D A; Harvey, R C; Faust, J B; Melnyk, O; Carey, K; Meeker, T C

1991-01-01

The t(11;14)(q13;q32) translocation has been associated with human B-lymphocytic malignancy. Several examples of this translocation have been cloned, documenting that this abnormality joins the immunoglobulin heavy-chain gene to the bcl-1 locus on chromosome 11. However, the identification of the bcl-1 gene, a putative dominant oncogene, has been elusive. In this work, we have isolated genomic clones covering 120 kb of the bcl-1 locus. Probes from the region of an HpaII-tiny-fragment island identified a candidate bcl-1 gene. cDNAs representing the bcl-1 mRNA were cloned from three cell lines, two with the translocation. The deduced amino acid sequence from these clones showed bcl-1 to be a member of the cyclin gene family. In addition, our analysis of expression of bcl-1 in an extensive panel of human cell lines showed it to be widely expressed except in lymphoid or myeloid lineages. This observation may provide a molecular basis for distinct modes of cell cycle control in different mammalian tissues. Activation of the bcl-1 gene may be oncogenic by directly altering progression through the cell cycle. Images PMID:1833629

MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

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

Qin, Bing; Xiao, Bo; Liang, Desheng

Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth,more » proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the expression of Bcl-2, suggesting potential novel therapeutic targets for atherosclerosis.« less

BCL-2 and BCL-XL expression are down-regulated in benign prostate hyperplasia nodules and not affected by finasteride and/or celecoxib

PubMed Central

Li, Feng; Pascal, Laura E; Zhou, Jianhua; Zhou, Yibin; Wang, Ke; Parwani, Anil V; Dhir, Rajiv; Guo, Peng; He, Dalin; Nelson, Joel B; Wang, Zhou

2018-01-01

The mechanisms involved in the development of benign prostatic hyperplasia (BPH) are poorly understood. One potential mechanism involved in BPH pathogenesis may involve altered expression of genes related to apoptosis and proliferation because reduced cell death and increased proliferation are thought to contribute to prostatic enlargement. This study examined the expression of B-cell lymphoma 2 (BCL-2) and B-cell lymphoma-extra large (BCL-XL), two important anti-apoptosis factors that are also capable of inhibiting cell proliferation via accelerated G1 arrest or delayed G1/S transition, using immunostaining in simple prostatectomy BPH specimens from patients naïve to androgen manipulation. Since androgens and inflammation are thought to play important roles in BPH pathogenesis, we tested the effect of inhibiting 5a-reductase and/or COX-2 on the expression of BCL-2 and BCL-XL in BPH specimens from prostate cancer patients with BPH. These patients had no prior use of chronic NSAIDs and/or 5a-reductase inhibitors and were treated with celecoxib, finasteride, celecoxib plus finasteride or no treatment for 28 consecutive days prior to surgery. In all specimens, BCL-2 and BCL-XL staining was evident in both luminal and basal epithelial cells, with more intense staining in basal cells. Both luminal and basal cells exhibited decreased BCL-2 and BCL-XL staining in BPH nodules compared to the surrounding normal prostatic tissues. In prostate cancer patients with BPH, celecoxib and/or finasteride did not affect the expression of BCL-2 and BCL-XL in luminal or basal cells in BPH nodules and normal adjacent tissues. These results suggest that BCL-2 and BCL-XL may act as anti-proliferative factors in BPH pathogenesis, and the effect of celecoxib and/or finasteride on BPH is unlikely mediated through modulating BCL-2 and BCL-XL signaling. PMID:29531971

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.

Rituximab-induced inhibition of YY1 and Bcl-xL expression in Ramos non-Hodgkin's lymphoma cell line via inhibition of NF-kappa B activity: role of YY1 and Bcl-xL in Fas resistance and chemoresistance, respectively.

PubMed

Vega, Mario I; Jazirehi, Ali R; Huerta-Yepez, Sara; Bonavida, Benjamin

2005-08-15

Rituximab treatment of B non-Hodgkin's lymphoma (NHL) cell lines inhibits the constitutive NF-kappaB activity and results in the sensitization of tumor cells to both chemotherapy and Fas-induced apoptosis. Cells expressing dominant active IkappaB or treated with NF-kappaB-specific inhibitors were sensitive to both drugs and Fas agonist mAb (CH-11)-induced apoptosis. Down-regulation of Bcl-xL expression via inhibition of NF-kappaB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL-overexpressing Ramos cells, Ramos hemagglutinin (HA)-Bcl-x, which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent; Ramos HA-Bcl-x cells were as sensitive as the wild type to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor yin-yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab or the NO donor DETANONOate or after transfection with YY1 small interfering RNA resulted in up-regulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two mechanisms underlying the chemosensitization and immunosensitization of B-NHL cells by rituximab via inhibition of NF-kappaB. The regulation of chemoresistance by NF-kappaB is mediated via Bcl-xL expression, whereas the regulation of Fas resistance by NF-kappaB is mediated via YY1 expression and activity. The potential clinical significance of these findings is discussed.

PB-1: The Relationship Between Anti Apoptotic Marker (BCL-2) and Biochemical Markers in Type 2 Diabetes Patients

PubMed Central

Damitri, TD; Faridah, AR; Imran, Y; Hasnan, J.

2006-01-01

Purpose : To investigate the expression of anti apoptotic marker (bcl-2) and the level of biochemical markers in type 2 diabetes patients. METHODS : A cross-sectional study was conducted from August 2003 to November 2005. Forty one type 2 diabetes patients and 36 non diabetes (control) subjects aged between 20 to 70 years were included in this study. Blood samples were collected for fasting plasma glucose (FPG), triglycerides (TG), Total cholesterol (TC), High density lipoprotein cholesterol (HDLC), Low density lipoprotein cholesterol (LDLC) and analyzed in the Chemical Pathology laboratory, while glycosylated hemoglobin A1c (A1C) was analyzed in the Endocrine laboratory. The skin biopsy tissue samples were stained with immunohistochemistry (IHC) stain for expression of bcl-2 in the Pathology laboratory. RESULTS : There was a significant difference (p<0.001) between both groups for mean FPG (diabetics=11.02±4.25, control=4.41±1.12 mmol/L), HDLC (diabetics=1.00±0.38, control=1.47±0.72 mmol/L) and A1C (diabetics=9.50±2.24%, control=5.00±0.67%). However, there was no significant difference for TG, TC, and LDLC between both groups. Interestingly, the difference of mean bcl-2 expression were very highly significant (p<0.001) when compared between both groups. Mean bcl-2 expression was dibetics= 1.88±0.33 and control= 1.47±0.51. Positive bcl-2 expression was found in only 5 (12.2%) diabetics while 36 (87.8%) diabetics showed negative expression. Positive bcl-2 expression was observed in 19 (52.8%) controls while 17 (47.2%) showed negative expression. CONCLUSION : The expression of anti apoptotic marker bcl-2 was increased in non diabetic subjects in order to prevent cell death. However, the reduced expression of bcl-2 in diabetic patients may be associated with programmed cell death. The detailed mechanism for the gene expression of bcl-2 may help us to understand how bcl-2 is involved in apoptosis in diabetic microvasculature complications.

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.

Expression of Bcl-2 in canine osteosarcoma

PubMed Central

Piro, F.; Leonardi, L.

2015-01-01

Osteosarcoma (OS) is the most common primary malignancy of bone. It is responsible for 80-85% of the primary bone tumors affecting dogs and it is characterized by aggressive and invasive behavior, with a high metastatic potential. Several studies on cancer and related tumorigenesis, show an involvement of the mechanisms of programmed cell death and cell survival. Many signals seem to be involved in the related mechanism of autophagy and in particular, our interest is focused on the expression of a family of Bcl-2 that seems to be involved either in the control of biomolecular mechanisms like autophagy and apoptosis. In this study we investigated the expression of Bcl-2 in different cases of spontaneous canine osteosarcoma and the related preliminary results are described. We found Bcl-2 activity was increased in OS tissue compared to normal bone tissue. These results suggested that Bcl-2 activity may play an important role in the formation of OS and as a diagnostic for neoplastic activity. However, further research is needed to confirm the role of Bcl-2 activity in OS in canines. PMID:26623359

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.

Signet-ring cell lymphoma: clinicopathologic, immunohistochemical, and fluorescence in situ hybridization studies of 7 cases.

PubMed

Zhang, Shanxiang; Sun, Jihong; Fang, Yanan; Nassiri, Mehdi; Liu, Lanting; Zhou, Jiehao; Stohler, Ryan; Choi, Haki; Vance, Gail H

2017-02-01

Signet-ring cell lymphoma (SRCL) is a rare morphologic variant of non-Hodgkin lymphoma. Although it was initially reported as a rare morphologic variant of follicular lymphoma (FL), SRCL has to date been described in most types of non-Hodgkin lymphoma, mostly as single-case reports. To study SRCL systematically by immunohistochemical stains and fluorescent in situ hybridization analyses. Seven SRCL cases were stained for CD3, CD5, CD20, PAX-5, CD10, CD21, CD23, cyclin D1, BCL2, BCL6, Ki-67, and MUM-1, and were analyzed by fluorescent in situ hybridization for BCL2, BCL6, MYC, and MALT1 rearrangements. Clinical information and patient outcome were reviewed in all patients. The patients were 3 women and 3 men, ranging in age from 31 to 75 years (average 60.3 years). The lesions involved lymph nodes, tonsil, parotid gland, soft tissue, and breast. There were 4 FLs, 1 diffuse large B-cell lymphoma (DLBCL), 1 DLBCL with FL, and 1 DLBCL with marginal zone lymphoma. All cases had typical signet-ring cell morphology. They were positive for CD20 and BCL-2, and had low-to-intermediate Ki-67 proliferation index (10%-40%) except in the parotid DLBCL with FL (70%). BCL-6 was detected in all but 1 FL (6/7). Fluorescent in situ hybridization detected IGH/BCL2 translocation in 1 FL, increased BCL6 copy number in another FL, BCL6 rearrangement, and increased copy number of MYC and MALT1 in the DLBCL with marginal zone lymphoma. The FL with signet-ring cell morphology (1/5) tends to lack IGH/BCL2 translocation, and an extended immunohistochemical study is recommended for correct diagnosis and classification of SRCL. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a B lymphoid disorder defined by specific morphologic features, a del(11)(q13) and a same breakpoint far from CCND1

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

Morel, D.; Callet, E.; Reynaud, S.

Chromosome rearrangements in 11q13 have been shown to occur in a variety of diffuse small B-cell lymphomas/leukemias, including, beside mantle cell lymphomas (MCL), some cases of CLL/SLL, PLL, and SLVL. If t(11;14)(q13;q32) may be considered as a hallmark of MCL, less is known about deletions involving 11q13. A series of 13 patients with a diffuse small B-lymphoma/leukemia was examined for morphology (cytology and histology), immunology, cytogenetics and FISH for some of them. According to karyotype findings, 2 groups were identified: Group 1: 9 patients (6M,3F), median age 60 yrs. without 11q anomalies. Apart from trisomy 12 (3 cases), diverse anomaliesmore » were identified including chromosomes 1, 2, 7, 8, 12, 17. Cases were classified as CLL (2), SLL/SLP (5), blast-rich immunocytomas (2). Group 2: 4 patients, all males, median age 52 yrs. with breakpoints in 11q13; there were 3 deletions, and a t(11;14) was present in another case. 2 patients presented with refractory disease followed for 23 and 9 months, respectively, without any consistent morphologic change, the chromosomal anomaly being present at diagnosis in 1.3 cases. Cytologically, a nucleolated cell component was the constant and striking feature and FISH study by cos 14, pHS 11, cos 17, cos 105 revealed the same breakpoint located far from CCND1. The fourth case, bearing the t(11;14), was diagnosed as CLL/PLL in cytology, but was histologically consistent with MCL; the breakpoint was located by FISH into the BCL1 locus. Even if this study needs further confirmation, it points at the 11q13 deletion as a genetic event leading to a more aggressive disease, associated with distinct cytologic features differing from MCL and a molecular event probably not involving BCL1/CCND1.« less

Proteomic study reveals a co-occurrence of gallic acid-induced apoptosis and glycolysis in B16F10 melanoma cells.

PubMed

Liu, Cheng; Lin, Jen-Jie; Yang, Zih-Yan; Tsai, Chi-Chu; Hsu, Jue-Liang; Wu, Yu-Jen

2014-12-03

Gallic acid (GA) has long been associated with a wide range of biological activities. In this study, its antitumor effect against B16F10 melanoma cells was demonstrated by MTT assay, cell migration assay, wound-healing assay, and flow cytometric analysis. GA with a concentration >200 μM shows apoptotic activity toward B16F10 cells. According to Western blotting data, overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptotic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-xL indicate that GA induces B16F10 cell apoptosis via mitochondrial pathway. The 2-DE based comparative proteomics was further employed in B16F10 cells with and without GA treatment for a large-scale protein expression profiling. A total of 41 differential protein spots were quantified, and their identities were characterized using LC-MS/MS analysis and database matching. In addition to some regulated proteins that were associated with apoptosis, interestingly, some identified proteins involved in glycolysis such as glucokinase, α-enolase, aldolase, pyruvate kinase, and GAPDH were simultaneously up-regulated, which reveals that the GA-induced cellular apoptosis in B16 melanoma cells is associated with metabolic glycolysis.

Exercise induces autophagy in peripheral tissues and in the brain.

PubMed

He, Congcong; Sumpter, Rhea; Levine, Beth

2012-10-01

We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We found that BCL2 AAA mice could not run on a treadmill as long as wild-type mice, and did not undergo exercise-mediated increases in skeletal glucose muscle uptake. Unlike wild-type mice, the BCL2 AAA mice failed to reverse high-fat diet-induced glucose intolerance after 8 weeks of exercise training, possibly due to defects in signaling pathways that regulate muscle glucose uptake and metabolism during exercise. Together, these findings suggested a hitherto unknown important role of autophagy in mediating exercise-induced metabolic benefits. In the present addendum, we show that treadmill exercise also induces autophagy in the cerebral cortex of adult mice. This observation raises the intriguing question of whether autophagy may in part mediate the beneficial effects of exercise in neurodegeneration, adult neurogenesis and improved cognitive function.

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.

Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets

PubMed Central

Marchetto, Aruna; Gerke, Julia S.; Rubio, Rebeca Alba; Kiran, Merve M.; Musa, Julian; Knott, Maximilian M. L.; Ohmura, Shunya; Li, Jing; Akpolat, Nusret; Akatli, Ayse N.; Özen, Özlem; Dirksen, Uta; Hartmann, Wolfgang; de Alava, Enrique; Baumhoer, Daniel; Sannino, Giuseppina; Kirchner, Thomas; Grünewald, Thomas G. P.

2018-01-01

Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care. PMID:29416716

Design, synthesis and characterization of novel quinacrine analogs that preferentially kill cancer over non-cancer cells through the down-regulation of Bcl-2 and up-regulation of Bax and Bad.

PubMed

Solomon, V Raja; Almnayan, Danah; Lee, Hoyun

2017-09-08

Both quinacrine, which contains a 9-aminoacridine scaffold, and thiazolidin-4-one are promising anticancer leads. In an attempt to develop effective and potentially safe anticancer agents, we synthesized 23 novel hybrid compounds by linking the main structural unit of the 9-aminoacridine ring with the thiazolidin-4-one ring system, followed by examination of their anticancer effects against three human breast tumor cell lines and matching non-cancer cells. Most of the hybrid compounds showed good activities, and many of them possessed the preferential killing property against cancer over non-cancer cells. In particular, 3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-thiazolidin-4-one (11; VR118) effectively killed/inhibited proliferation of cancer cells at IC 50 values in the range of 1.2-2.4 μM. Furthermore, unlike quinacrine or cisplatin, compound 11 showed strong selectivity for cancer cell killing, as it could kill cancer cells 7.6-fold (MDA-MB231 vs MCF10A) to 14.7-fold (MCF7 vs MCF10A) more effectively than matching non-cancer cells. Data from flow cytometry, TUNEL and Western blot assays showed that compound 11 kills cancer cells by apoptosis through the down-regulation of Bcl-2 (but not Bcl-X L ) survival protein and up-regulation of Bad and Bax pro-apoptotic proteins. Thus, compound 11 is a highly promising lead for an effective and potentially anticancer therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

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.

[Diffuse large B-cell lymphoma with concomitant c-MYC and BCL6 gene rearrangements with primary skin involvement: A case report and a review of literature].

PubMed

Gabeeva, N G; Koroleva, D A; Belyaeva, A V; Chernova, N G; Kuzmina, L A; Sudarikov, A B; Obukhova, T N; Kovrigina, A M; Zvonkov, E E; Savchenko, V G

Double-hit lymphoma (DHL) is a rare aggressive B-cell lymphoma with concomitant c-MYC, BCL2 or BCL6 gene rearrangements, which is characterized by the high frequency of extranodal lesions and by resistance to chemotherapy. The median survival does not exceed 18 months in patients with this disease. The majority of DHL is represented by с-MYC/BCL2 cases. The combination of c-MYC/BCL6 occurs rarely (5-8%). The paper describes a case of DHL with concomitant c-MYC and BCL6 gene rearrangements, which mimics diffuse large B-cell lymphoma, leg-type.

Involvement of p53 and Bcl-2 in sensory cell degeneration in aging rat cochleae.

PubMed

Xu, Yang; Yang, Wei Ping; Hu, Bo Hua; Yang, Shiming; Henderson, Donald

2017-06-01

p53 and Bcl-2 (B-cell lymphoma 2) are involved in the process of sensory cell degeneration in aging cochleae. To determine molecular players in age-related hair cell degeneration, this study examined the changes in p53 and Bcl-2 expression at different stages of apoptotic and necrotic death of hair cells in aging rat cochleae. Young (3-4 months) and aging (23-24 months) Fisher 344/NHsd rats were used. The thresholds of the auditory brainstem response (ABR) were measured to determine the auditory function. Immunolabeling was performed to determine the expression of p53 and Bcl-2 proteins in the sensory epithelium. Propidium iodide staining was performed to determine the morphologic changes in hair cell nuclei. Aging rats exhibited a significant elevation in ABR thresholds at all tested frequencies (p < 0.001). The p53 and Bcl-2 immunoreactivity was increased in aging hair cells showing the early signs of apoptotic changes in their nuclei. The Bcl-2 expression increase was also observed in hair cells displaying early signs of necrosis. As the hair cell degenerative process advanced, p53 and Bcl-2 immunoreactivity became reduced or absent. In the areas where no detectable nuclear staining was present, p53 and Bcl-2 immunoreactivity was absent.

Up-Regulation of Bcl-xl by Hepatocyte Growth Factor in Human Mesothelioma Cells Involves ETS Transcription Factors

PubMed Central

Cao, Xiaobo; Littlejohn, James; Rodarte, Charles; Zhang, Lidong; Martino, Benjamin; Rascoe, Philip; Hamid, Kamran; Jupiter, Daniel; Smythe, W. Roy

2009-01-01

Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl. PMID:19834061

Association of BCL11A genetic variant (rs11886868) with severity in β-thalassaemia major & sickle cell anaemia.

PubMed

Dadheech, Sneha; Madhulatha, D; Jainc, Suman; Joseph, James; Jyothy, A; Munshi, Anjana

2016-04-01

The amount of foetal haemoglobin that persists in adulthood affects the clinical severity of haemoglobinopathies including β-thalassaemia major and sickle cell anaemia (SCA). The present study was undertaken to analyse β-thalassaemia as well as SCA patients for the single nucleotide polymorphism (SNP), rs11886868 (T/C) in BCL11A gene and to evaluate the association between this polymorphism and severity of β-thalassaemia major and SCA. a total of 620 samples (420 β-thalassaemia major and 200 SCA cases) were analysed before blood transfusion using basic screening tests like complete blood analysis and osmotic fragility and further confirmed by high performance liquid chromatography (HPLC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot blot techniques. All patients were transfusion dependent. Patients with β-thalassaemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. β-thalassaemia as well as SCA patients were analysed for the SNP, rs11886868 (T/C) in BCL11A gene and association between this polymorphism and severity of β-thalassaemia major as well as SCA was evaluated. There was a significant difference in genotypic and allelic frequencies of BCL11A gene polymorphism between mild and moderate and mild and severe cases in both the groups. A significant (P<0.001) difference was observed in the mean HbF levels between the three genotypes in different severity groups. HbF levels were found to be high in CC genotype bearing individuals followed by TC and TT in β-thalassaemia major as well as SCA. This study confirms that the T/C variant (rs11886868) of the BCL11A gene causing downregulation of BCL11A gene expression in adult erythroid precursors results in the induction of HbF and ameliorates the severity of β-thalassaemia as well as SCA.

Analysis of alterations of oncogenes and tumor suppressor genes in chronic lymphocytic leukemia.

PubMed Central

Gaidano, G.; Newcomb, E. W.; Gong, J. Z.; Tassi, V.; Neri, A.; Cortelezzi, A.; Calori, R.; Baldini, L.; Dalla-Favera, R.

1994-01-01

B cell chronic lymphocytic leukemia (B-CLL) represents the most frequent adult leukemia in the Western world. The molecular pathogenesis of B-CLL is largely unknown. Although initial reports on small panels of cases had suggested a role for Bcl-1 and Bcl-2 oncogene activation in B-CLL, later investigations failed to confirm these data. Among tumor suppressor genes, p53 mutations have been reported in a fraction of cases. In this study, we have attempted a conclusive definition of the involvement of dominantly acting oncogenes (Bcl-1 and Bcl-2) and tumor suppressor loci (p53, 6q-) in 100 cases of B-CLL selected for their CD5 positivity and Rai's stage (0 to IV). Rearrangements of Bcl-1 and Bcl-2 and deletions of 6q and 17p were analyzed by Southern blot using multiple probes. Mutational analysis (single strand conformation polymorphism and polymerase chain reaction direct sequencing) was used to assay p53 inactivation. No alterations of Bcl-1 or Bcl-2 were detected in the 100 cases tested. Mutations of p53 were found in 10/100 cases without any significant association with clinical stage. Deletions of 6q were present in 4/100 cases. Overall, our data indicate that: 1) contrary to previous reports, Bcl-1 and Bcl-2 rearrangements are not involved in CD5+ B-CLL pathogenesis and 2) p53 mutations are present in 10% of cases at all stages of the disease. Images Figure 1 Figure 2 Figure 3 PMID:8203469

Neoadjuvant docetaxel treatment for locally advanced prostate cancer: a clinicopathologic study.

PubMed

Magi-Galluzzi, Cristina; Zhou, Ming; Reuther, Alwyn M; Dreicer, Robert; Klein, Eric A

2007-09-15

The objective of the current study was to determine the histologic and molecular changes that occurred in patients with high-risk, localized prostate cancer (PCa) after neoadjuvant docetaxel chemotherapy. Patients who had locally advanced PCa (serum preoperative [initial] prostate-specific antigen [iPSA] level >or=15 ng/mL, or clinical >or=T2b disease, or biopsy Gleason score [GS] >or=8) and no evidence of metastatic disease received 6 doses of intravenous docetaxel (40 mg/m(2)) administered weekly for 6 weeks followed by radical prostatectomy (RP). The Wilcoxon signed-rank test was used to compare pretreatment and posttreatment markers. Twenty-eight patients completed chemotherapy and underwent RP at the Cleveland Clinic; none achieved a pathologic complete response. Pretreatment diagnostic prostate biopsies (PBx) were reviewed in all patients, and unstained sections of formalin-fixed tissue were available from 11 patients. The median patient age was 62 years (range, 49-72 years), and the median iPSA was 6.8 ng/mL (range, 2.5-24 ng/mL). At a median follow-up of 49.5 months (range, 23-72 months), 12 patients (43%) remained clinically and biochemically free of disease with no additional therapy, and 16 patients (57%) had biochemical failure. The pretreatment GS was 6 in 2 patients (7%), 7 in 10 patients (36%), 8 in 11 patients (39%) and 9 in 5 patients (18%). Two patients (7.1%) had organ-confined disease, and 23 patients (82.1%) had extraprostatic extension. Four patients (14.3%) had positive lymph nodes, and 11 patients (39.3%) had seminal vesicle involvement. Immunohistochemical (IHC) staining for a panel of markers involved in various cellular functions (alpha-methylacyl-coenzyme A racemase [AMACR], beta-tubulin I, beta-tubulin III, cyclin D1, p27, p21, Ki-67, p53, Bcl-2, and an apoptosis detection kit [ApopTag]) was performed on a tissue microarray that contained the posttreatment (RP) tissue specimens and on the PBx specimens, if available. When the IHC staining patterns were compared between PBx and RP specimens using the Wilcoxon signed-rank test, only p53 expression (P = .017) and Bcl-2 expression (P = .014) were found to be increased significantly after neoadjuvant docetaxel treatment. However, after performing the Bonferroni adjustment, these differences were no longer significant (P > .005). Ki-67, ApopTag, beta-tubulin I, and beta-tubulin III expression levels also were increased after treatment; however, the differences were not found to be statistically significant. The expression levels of AMACR, p27, p21, and cyclin D1 were comparable in pretreatment and posttreatment specimens. The current results indicated that, although it will require longer follow-up studies and larger numbers of patients to ascertain the value of neoadjuvant treatment, the negative findings of the current study may explain the lack of clinical response in patients who received neoadjuvant docetaxel for PCa. Although the results were subject to interpretation limits based on the study size, the increased expression of p53 and Bcl-2 that was detected after treatment using the Wilcoxon signed-rank test suggested that the apoptotic pathway may be an important target for this drug, and further investigation is warranted. (c) 2007 American Cancer Society.

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 predictors of OS, whereas negative BCL2 expression, lymph node involvement, and high-stage disease were independent predictors of DFS. Compared with low- and moderate-grade endometrioid ECs, non-endometrioid and high-grade endometrioid ECs showed significant DFF40 and BCL2 underexpression. The absence of DFF40 and BCL2 expression negatively affects DFS and OS. Further prospective studies are warranted to assess the potential utility of DFF40 and BCL2 as targets in the diagnosis or treatment of ECs.

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

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

Ji Lili; Shanghai R and D Centre for Standardization of Traditional Chinese Medicines, Shanghai 201203; Chen Ying

2008-09-15

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability.more » Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.« less

Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma

USDA-ARS?s Scientific Manuscript database

Naïve CD4+ T-helper cells differentiate into Th2 effector cells during asthma and helminth (worm) infection. Here we report that mice lacking the transcription factor Bcl11b in mature CD4+ T-cells are incapable of mounting an effective Th2 response in asthma and worm infection with a major reductio...

microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma

PubMed Central

Pal, Rekha; Greene, Stephanie

2015-01-01

This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR). Two medulloblastoma cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5) and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival. PMID:26394044

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

A New Gene Expression Signature for Triple-Negative Breast Cancer Using Frozen Fresh Tissue before Neoadjuvant Chemotherapy

PubMed Central

Santuario-Facio, Sandra K; Cardona-Huerta, Servando; Perez-Paramo, Yadira X; Trevino, Victor; Hernandez-Cabrera, Francisco; Rojas-Martinez, Augusto; Uscanga-Perales, Grecia; Martinez-Rodriguez, Jorge L; Martinez-Jacobo, Lizeth; Padilla-Rivas, Gerardo; Muñoz-Maldonado, Gerardo; Gonzalez-Guerrero, Juan Francisco; Valero-Gomez, Javier; Vazquez-Guerrero, Ana L; Martinez-Rodriguez, Herminia G; Barboza-Quintana, Alvaro; Barboza-Quintana, Oralia; Garza-Guajardo, Raquel; Ortiz-Lopez, Rocio

2017-01-01

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer tumors. Comparisons between TNBC and non–triple-negative breast cancer (nTNBC) may help to differentiate key components involved in TNBC neoplasms. The purpose of the study was to analyze the expression profile of TNBC versus nTNBC tumors in a homogeneous population from northeastern Mexico. A prospective study of 50 patients (25 TNBC and 25 nTNBC) was conducted. Clinic parameters were equally distributed for TNBC and nTNBC: age at diagnosis (51 versus 47 years, p = 0.1), glucose level (107 mg/dl versus 104 mg/dl, p = 0.64), and body mass index (28 versus 29, p = 0.14). Core biopsies were collected for histopathological diagnosis and gene expression analysis. Total RNA was isolated and expression profiling was performed. Forty genes showed differential expression pattern in TNBC tumors. Among these, nine overexpressed genes (PRKX/PRKY, UGT8, HMGA1, LPIN1, HAPLN3, FAM171A1, BCL141A, FOXC1, and ANKRD11), and one underexpressed gene (ANX9) are involved in general metabolism. Based on this biochemical peculiarity and the overexpression of BCL11A and FOXC1 (involved in tumor growth and metastasis, respectively), we validated by quantitative polymerase chain reaction the expression profiles of seven genes out of the signature. In this report, a new gene signature for TNBC is proposed. To our knowledge, this is the first TNBC signature that describes genes involved in general metabolism. The findings may be pertinent for Mexican patients and require evaluation in other ethnic groups and populations. PMID:28474731

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.

Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation.

PubMed

Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B; Lenz, Georg; Ruland, Jürgen

2015-12-29

The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL.

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.

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

Xp11.2 translocation renal cell carcinoma occurring during pregnancy with a novel translocation involving chromosome 19: a case report with review of the literature

PubMed Central

Armah, Henry B; Parwani, Anil V; Surti, Urvashi; Bastacky, Sheldon I

2009-01-01

The recently recognized renal cell carcinomas (RCCs) associated with Xp11.2 translocations (TFE3 transcription factor gene fusions) are rare tumors predominantly reported in children. They comprise at least one-third of pediatric RCCs and only few adult cases have been reported. Here, we present a case of Xp11.2 translocation RCC in 26-year-old pregnant female. Her routine antenatal ultrasonography accidentally found a complex cystic right renal mass. Further radiologic studies revealed unilocular cyst with multiple mural nodules at inferior pole of right kidney, which was suspicious for RCC. She underwent right radical nephrectomy at 15 weeks gestation. Macroscopically, the cystic tumor was well encapsulated with multiple friable mural nodules on its inner surface. Microscopically, the tumor consisted of clear and eosinophilic/oncocytic voluminous cells arranged in papillary, trabecular, and nested/alveolar patterns. Occasional hyaline nodules and numerous psammoma bodies were present. Immunohistochemically, the tumor showed strong nuclear positivity for TFE3. Epithelial membrane antigen, CD10, and E-cadherin were strongly positive. Cytokeratin AE1/AE3, cytokeratin CAM-5.2, calveolin, and parvalbumin were moderately positive. Cytokeratin 7, renal cell carcinoma antigen, and colloidal iron were focally weakly positive. BerEP4 and carbonic anhydrase IX were negative. Cytogenetically, the tumor harbored a novel variant translocation involving chromosomes X and 19, t(X;19)(p11.2;q13.1). Interphase FISH analysis performed on cultured and uncultured tumor cells using a dual-color break-apart DNA probe within the BCL3 gene on 19q13.3 was negative for the BCL3 gene rearrangement. She received no adjuvant therapy, delivered a normal term baby five months later, and is alive without evidence of disease 27 months after diagnosis and surgery. Unlike most recently reported Xp11.2 translocation RCCs in adult patients with aggressive clinical course, this adult case occurring during pregnancy with a novel translocation involving chromosome 19 followed an indolent clinical course. PMID:19450277

Xp11.2 translocation renal cell carcinoma occurring during pregnancy with a novel translocation involving chromosome 19: a case report with review of the literature.

PubMed

Armah, Henry B; Parwani, Anil V; Surti, Urvashi; Bastacky, Sheldon I

2009-05-18

The recently recognized renal cell carcinomas (RCCs) associated with Xp11.2 translocations (TFE3 transcription factor gene fusions) are rare tumors predominantly reported in children. They comprise at least one-third of pediatric RCCs and only few adult cases have been reported. Here, we present a case of Xp11.2 translocation RCC in 26-year-old pregnant female. Her routine antenatal ultrasonography accidentally found a complex cystic right renal mass. Further radiologic studies revealed unilocular cyst with multiple mural nodules at inferior pole of right kidney, which was suspicious for RCC. She underwent right radical nephrectomy at 15 weeks gestation. Macroscopically, the cystic tumor was well encapsulated with multiple friable mural nodules on its inner surface. Microscopically, the tumor consisted of clear and eosinophilic/oncocytic voluminous cells arranged in papillary, trabecular, and nested/alveolar patterns. Occasional hyaline nodules and numerous psammoma bodies were present.Immunohistochemically, the tumor showed strong nuclear positivity for TFE3. Epithelial membrane antigen, CD10, and E-cadherin were strongly positive. Cytokeratin AE1/AE3, cytokeratin CAM-5.2, calveolin, and parvalbumin were moderately positive. Cytokeratin 7, renal cell carcinoma antigen, and colloidal iron were focally weakly positive. BerEP4 and carbonic anhydrase IX were negative. Cytogenetically, the tumor harbored a novel variant translocation involving chromosomes X and 19, t(X;19)(p11.2;q13.1). Interphase FISH analysis performed on cultured and uncultured tumor cells using a dual-color break-apart DNA probe within the BCL3 gene on 19q13.3 was negative for the BCL3 gene rearrangement. She received no adjuvant therapy, delivered a normal term baby five months later, and is alive without evidence of disease 27 months after diagnosis and surgery. Unlike most recently reported Xp11.2 translocation RCCs in adult patients with aggressive clinical course, this adult case occurring during pregnancy with a novel translocation involving chromosome 19 followed an indolent clinical course.

Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1

PubMed Central

Maiuri, M Chiara; Le Toumelin, Gaëtane; Criollo, Alfredo; Rain, Jean-Christophe; Gautier, Fabien; Juin, Philippe; Tasdemir, Ezgi; Pierron, Gérard; Troulinaki, Kostoula; Tavernarakis, Nektarios; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-01-01

The anti-apoptotic proteins Bcl-2 and Bcl-XL bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114–123). The physical interaction between Beclin-1 and Bcl-XL is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-XL is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-XL abolishes the Bcl-XL-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-XL, antagonizes autophagy inhibition by Bcl-2/Bcl-XL and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-XL. PMID:17446862

[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.

The protective efficacy and safety of bandage contact lenses in children aged 5 to 11 after frontalis muscle flap suspension for congenital blepharoptosis: A single-center randomized controlled trial.

PubMed

Chen, Lin; Pi, Lianhong; Ke, Ning; Chen, Xinke; Liu, Qing

2017-09-01

Postoperative complications, lagophthalmos and exposure keratopathy sometimes occur after surgery for congenital blepharoptosis. Bandage contact lenses (BCL) can help prevent some ocular surface disorders. The study aims to evaluate the efficacy and safety of BCL for protection of the ocular surface in children aged 5 to 11 years after frontalis muscle flap suspension for congenital blepharoptosis. We conducted a prospective randomized clinical study of 30 eyes of 30 patients with congenital blepharoptosis consecutively enrolled at the Ophthalmology Ward of the Children's Hospital of Chongqing Medical University, China from September 1, 2016, to February 30, 2017. After frontalis muscle flap suspension surgery, patients were randomly assigned to undergo BCL application (BCL group, 15 eyes) or no BCL application (control group, 15 eyes). All patients were treated with bramycin 0.3% and polyvinyl alcohol drops after surgery. The primary outcomes were dry eye assessed by tear film break time (TFBUT), fluoresce in corneal staining (FCS) on slit-lamp on days 1, 3, and 15 postoperatively, and lower tear meniscus height (LTMH) on optical coherence tomography on days 1 and 15 postoperatively. Secondary outcomes were pairwise correlation of TFBUT, FCS and LTMH. In the BCL group, abnormal TFBUT and FCS were only found in 2 patients (13.33%) on postoperative day 15. In the control group, the incidence of dry eye assessed by TFBUT was 67.00% (10/15 eyes) on day 1, 73.33% (11/15 eyes) on day 3, and 53.33% (8/15 eyes) on day 15 (P < .001). LTMH were significantly higher in the BCL group than the control group postoperatively (P < .001). Significant positive correlations were found between LTMH and TFBUT pre-operation and on days 1 and 15 post-operation. For LTMH and FCSS (R = -0.815, P < .001), and TFBUT and FCS (R = -0.837, P < .001), the Pearson coefficient was negative on postoperative day 1, but not correlated on day 15. Silicone hydrogel BCL were safe and efficacious for protective use in children after frontalis muscle flap suspension for congenital blepharoptosis.

Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

PubMed

Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

2009-12-01

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo

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

Cerchietti, L.C.; Ghetu, A.F.; Zhu, X.

2010-09-22

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compoundmore » also killed primary DLBCLs from human patients.« less

The effect of histone deacetylase inhibitors on AHSP expression

PubMed Central

Ziari, Katayoun; Ranjbaran, Reza; Nikouyan, Negin

2018-01-01

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies. PMID:29389946

Effects of mycobacteria on regulation of apoptosis in mononuclear phagocytes.

PubMed Central

Klingler, K; Tchou-Wong, K M; Brändli, O; Aston, C; Kim, R; Chi, C; Rom, W N

1997-01-01

Since apoptosis is observed in tuberculous granulomata, we investigated the molecular mechanisms underlying the apoptotic pathway in an in vitro model of mycobacterial infection of mononuclear phagocytes. We postulated that Mycobacterium tuberculosis could trigger the apoptotic pathway in macrophages, resulting in death of the microorganism by modulating the expression of bcl-2, bax, bcl-xL, and bcl-xS. We found that the mRNA of bcl-2, an inhibitor of apoptosis, was downregulated in peripheral blood monocytes (PBM) between 2 and 6 h following infection with M. bovis BCG or induction with heat-killed M. tuberculosis H37Ra. Western analysis showed a downregulation of the Bcl-2 protein, with a half-life of 24 h. At the same time points, there was no change in the expression of Bax or Bcl-xS, inducers of apoptosis, but Bcl-xL, another inhibitor of apoptosis, was minimally upregulated by BCG. To determine if apoptosis could be a mechanism for growth inhibition in vivo, we obtained alveolar macrophages by bronchoalveolar lavage from involved sites in patients with active pulmonary tuberculosis. Using the TUNEL (terminal deoxynucleotidyltransferase mediated nick end labeling) technique, we observed significantly more apoptosis in involved segments of five tuberculosis patients (14.8 +/- 1.9%) than in those of normal controls (<1%, P = 0.02) or in uninvolved segments (4.3 +/- 0.9%, P < 0.05). We conclude that apoptosis of mononuclear phagocytes induced by M. tuberculosis occurs in vivo and that in an in vitro model of mycobacterial infection, apoptosis may be mediated by downregulation of Bcl-2. PMID:9393826

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.

Ginkgo biloba leaf extract and alpha-tocopherol attenuate haloperidol-induced orofacial dyskinesia in rats: Possible implication of antiapoptotic mechanisms by preventing Bcl-2 decrease and Bax elevation.

PubMed

An, Hui Mei; Tan, Yun Long; Shi, Jing; Wang, Zhiren; Lv, Meng Han; Soares, Jair C; Zhou, Dongfeng; Yang, Fude; Zhang, Xiang Yang

2016-12-01

Tardive dyskinesia (TD) is a serious side effect of long-term administration of typical neuroleptics, such as haloperidol. The pathophysiology of TD remains unclear, but the experimental evidence suggests that free radical-induced neuronal apoptosis in the basal ganglia may play an important role. This study was to investigate changes in Bax and Bcl-2 expression levels in TD-associated brain regions and the effects of the antioxidant EGb761 on Bax and Bcl-2 levels in an animal model of TD. Thirty-two rats were randomly divided into four study groups: saline control (saline), haloperidol-alone (haloperidol), EGb761-haloperidol (EGb), and alpha-tocopherol-haloperidol (vitamin E). Rats were treated with daily intraperitoneal haloperidol injections (2 mg/kg/day) for 5 weeks. EGb761 (50 mg/kg/day) and alpha-tocopherol (20 mg/kg/day) were then administered for another 5 weeks during the withdrawal period. Behavioral assessments were performed, and Bax and Bcl-2 protein expression levels were immunohistochemically analyzed in four brain regions, including the prefrontal cortex, striatum, substantia nigra, and globus pallidum. We found that increased vacuous chewing movements (VCMs) were associated with increased proapoptotic Bax protein expression, decreased antiapoptotic Bcl-2 protein expression, and an increased Bax/Bcl-2 ratio. EGb761 and alpha-tocopherol treatment reversed the increase in VCMs, decreased Bax expression, increased Bcl-2 expression, and decreased the Bax/Bcl-2 ratio. These results demonstrate that long-term haloperidol administration may affect Bcl-2 protein family expression and promote neuronal apoptosis in the basal ganglia. In combination with their antioxidant capacity, EGb761 and alpha-tocopherol's antiapoptotic effects through Bcl-2 might account for the symptom improvement observed in haloperidol-induced TD rats. Copyright © 2016 Elsevier GmbH. All rights reserved.

The NF-κB regulator Bcl-3 and the BH3-only proteins Bim and Puma control the death of activated T cells

PubMed Central

Bauer, Anette; Villunger, Andreas; Labi, Verena; Fischer, Silke F.; Strasser, Andreas; Wagner, Hermann; Schmid, Roland M.; Häcker, Georg

2006-01-01

Apoptosis of activated T cells is critical for the termination of immune responses. Here we show that adjuvant-stimulated dendritic cells secrete cytokines that prime activated T cells for survival and analyze the roles of the NF-κB regulator Bcl-3 and the proapoptotic Bcl-2 family members Bim and Puma. Bcl-3 overexpression increased survival, and activated bcl-3−/− T cells died abnormally rapidly. Cytokines from adjuvant-stimulated dendritic cells induced Bcl-3, but survival through cytokine priming was Bcl-3-independent. Apoptosis inhibition by Bcl-3 involved blockade of Bim activation, because Bim was overactivated in Bcl-3-deficient cells, and Bcl-3 failed to increase survival of bim−/− T cells. However, adjuvants increased survival also in Bim-deficient T cells. This Bim-independent death pathway is at least in part regulated by Puma, as shown by analysis of puma−/− and noxa−/− T cells. IL-1, IL-7, and IL-15 primed T cells for survival even in the absence of Bim or Puma. Our data define interrelations and a Bim-independent pathway to activated T cell death. PMID:16832056

BCL11A Enhancer Haplotypes and Fetal Hemoglobin in Sickle Cell Anemia

PubMed Central

Sebastiani, P.; Farrell, J.J.; Alsultan, A.; Wang, S.; Edward, H. L.; Shappell, H.; Bae, H.; Milton, J. N.; Baldwin, C.T.; Al-Rubaish, A.M.; Naserullah, Z.; Al-Muhanna, F.; Alsuliman, A.; Patra, P. K.; Farrer, L.A.; Ngo, D.; Vathipadiekal, V.; Chui, D.H.K.; Al-Ali, A.K.; Steinberg, M.H.

2015-01-01

Background Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. Methods and Results Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts was approximately 6%. Conclusions Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF. PMID:25703683

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

PubMed

Kakkola, L; Denisova, O V; Tynell, J; Viiliäinen, J; Ysenbaert, T; Matos, R C; Nagaraj, A; Ohman, T; Kuivanen, S; Paavilainen, H; Feng, L; Yadav, B; Julkunen, I; Vapalahti, O; Hukkanen, V; Stenman, J; Aittokallio, T; Verschuren, E W; Ojala, P M; Nyman, T; Saelens, X; Dzeyk, K; Kainov, D E

2013-07-25

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

PubMed

Lee, Suk Kyoo; Lee, Gyun Min

2003-06-30

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.

Hydrogen Sulfide Attenuates the Recruitment of CD11b+Gr-1+ Myeloid Cells and Regulates Bax/Bcl-2 Signaling in Myocardial Ischemia Injury

PubMed Central

Zhang, Youen; Li, Hua; Zhao, Gang; Sun, Aijun; Zong, Nobel C.; Li, Zhaofeng; Zhu, Hongming; Zou, Yunzeng; Yang, Xiangdong; Ge, Junbo

2014-01-01

Hydrogen sulfide, an endogenous signaling molecule, plays an important role in the physiology and pathophysiology of the cardiovascular system. Using a mouse model of myocardial infarction, we investigated the anti-inflammatory and anti-apoptotic effects of the H2S donor sodium hydrosulfide (NaHS). The results demonstrated that the administration of NaHS improved survival, preserved left ventricular function, limited infarct size, and improved H2S levels in cardiac tissue to attenuate the recruitment of CD11b+Gr-1+ myeloid cells and to regulate the Bax/Bcl-2 pathway. Furthermore, the cardioprotective effects of NaHS were enhanced by inhibiting the migration of CD11b+Gr-1+ myeloid cells from the spleen into the blood and by attenuating post-infarction inflammation. These observations suggest that the novel mechanism underlying the cardioprotective function of H2S is secondary to a combination of attenuation the recruitment of CD11b+Gr-1+ myeloid cells and regulation of the Bax/Bcl-2 apoptotic signaling. PMID:24758901

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.

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

PubMed Central

Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

2015-01-01

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

PubMed

Lauf, Peter K; Alqahtani, Tariq; Flues, Karin; Meller, Jaroslaw; Adragna, Norma C

2015-01-01

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis. Copyright © 2015 the American Physiological Society.

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

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

Involvement of the bax and bcl-2 system in the induction of germ cell apoptosis is correlated with the time of reperfusion after testicular ischemia in a rat model.

PubMed

Sukhotnik, Igor; Voskoboinik, Katya; Lurie, Michael; Bejar, Yakov; Coran, Arnold G; Mogilner, Jorge G

2009-10-01

The objective of this study was to examine the relationship between time of reperfusion and bax/bcl-2-dependent germ cell apoptosis after testicular ischemia-reperfusion injury in the rat. In ischemic testis, bax/bcl-2 ratio did not change significantly, and the elevation of germ cell apoptosis was not marked; in the contralateral testis, germ cell apoptosis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion and was initiated by decreased bcl-2 messenger RNA levels and elevated bax/bcl-2 ratio within the first 6 hours of reperfusion.

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.

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2.

PubMed

Huang, R P; Fan, Y; Peng, A; Zeng, Z L; Reed, J C; Adamson, E D; Boynton, A L

1998-09-11

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

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.

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

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

PubMed

Wride, M A; Parker, E; Sanders, E J

1999-09-01

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis. Copyright 1999 Academic Press.

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.

Targeting the BH3-interacting domain death agonist to develop mechanistically unique antidepressants

PubMed Central

Malkesman, O; Austin, DR; Tragon, T; Henter, ID; Reed, JC; Pellecchia, M; Chen, G; Manji, HK

2011-01-01

The BH3-interacting domain death agonist (Bid) is a pro-apoptotic member of the B-cell lymphoma-2 (Bcl-2) protein family. Previous studies have shown that stress reduces levels of Bcl-2 in brain regions implicated in the pathophysiology of mood disorders, whereas antidepressants and mood stabilizers increase Bcl-2 levels. The Bcl-2 protein family has an essential role in cellular resilience as well as synaptic and neuronal plasticity and may influence mood and affective behaviors. This study inhibited Bid in mice using two pharmacological antagonists (BI-11A7 and BI-2A7); the selective serotonin reuptake inhibitor citalopram was used as a positive control. These agents were studied in several well-known rodent models of depression—the forced swim test (FST), the tail suspension test (TST), and the learned helplessness (LH) paradigm—as well as in the female urine sniffing test (FUST), a measure of sex-related reward-seeking behavior. Citalopram and BI-11A7 both significantly reduced immobility time in the FST and TST and attenuated escape latencies in mice that underwent the LH paradigm. In the FUST, both agents significantly improved duration of female urine sniffing in mice that had developed helplessness. LH induction increased the activation of apoptosis-inducing factor (AIF), a caspase-independent cell death constituent activated by Bid, and mitochondrial AIF expression was attenuated by chronic BI-11A7 infusion. Taken together, the results suggest that functional perturbation of apoptotic proteins such as Bid and, alternatively, enhancement of Bcl-2 function, is a putative strategy for developing novel therapeutics for mood disorders. PMID:21727899

Method for separating boron isotopes

DOEpatents

Rockwood, Stephen D.

1978-01-01

A method of separating boron isotopes .sup.10 B and .sup.11 B by laser-induced selective excitation and photodissociation of BCl.sub.3 molecules containing a particular boron isotope. The photodissociation products react with an appropriate chemical scavenger and the reaction products may readily be separated from undissociated BCl.sub.3, thus effecting the desired separation of the boron isotopes.

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

Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling.

PubMed

Koehler, Bruno Christian; Jassowicz, Adam; Scherr, Anna-Lena; Lorenz, Stephan; Radhakrishnan, Praveen; Kautz, Nicole; Elssner, Christin; Weiss, Johanna; Jaeger, Dirk; Schneider, Martin; Schulze-Bergkamen, Henning

2015-11-19

Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials. Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux. This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12. The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.

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

Intracellular Insulin-like Growth Factor-I Induces Bcl-2 Expression in Airway Epithelial Cells 1

PubMed Central

Chand, Hitendra S.; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S.; Randell, Scott H.; Tesfaigzi, Yohannes

2012-01-01

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and IGF-1 coincided with induced Bcl-2 expression compared to controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using shRNA showed that intracellular (IC)-IGF-1 was increasing Bcl-2 expression. Blocking EGFR or IGF-1R activation also suppressed IC-IGF-1, and abolished the Bcl-2 induction. Induced expression and co-localization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and EGFR pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

PubMed Central

Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

2016-01-01

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients. PMID:26842479

Up-regulation of Bcl-2 through hyperbaric pressure transfection of TGF-beta1 ameliorates ischemia-reperfusion injury in rat cardiac allografts.

PubMed

Grünenfelder, Jürg; Miniati, Douglas N; Murata, Seiichiro; Falk, Volkmar; Hoyt, E Grant; Robbins, Robert C

2002-02-01

Oxidative stress after ischemia-reperfusion of cardiac allografts leads to activation of cardiomyocytes and production of cytokines. Bcl-2, an inhibitor of the apoptotic pathway, also has strong antioxidant properties. Ischemia-reperfusion injury after transplantation leads to decreased bcl-2 and increased tumor necrosis factor (TNF)-alpha levels. Transforming growth factor (TGF)-beta1 is known to attenuate ischemia-reperfusion injury and inhibits apoptosis of myofibroblasts. We hypothesize that TGF-beta1, prevents bcl-2 cleavage and increased TNF-alpha production. Rat PVG donor hearts were heterotopically transplanted into ACI recipients. Donor hearts were procured and assigned to groups: (1) intracoronary TGF-beta1 (200 ng/ml) perfusion and pressure at 78 psi for 45 minutes (n = 4); (2) intracoronary TGF-beta1 perfusion and incubation for 45 minutes without pressure (n = 4), (3) saline perfusion and incubation for 45 minutes without pressure (n = 4). Hearts were procured 4 hours after transplantation and analyzed by reverse transcriptase-polymerase chain reaction for bcl-2 mRNA expression, ELISA for TNF-alpha, and for myeloperoxidase activity (MPO). Bcl-2 decreased in untreated animals (bcl-2:G3PDH ratio = 0.85 +/- 0.73 vs 1.16 +/- 0.11, not significant [NS]), whereas TNF-alpha increased to 669.99 +/- 127.09 vs 276.84 +/- 73.65 pg/mg total protein in controls (p < 0.003). In TGF-beta(1) pressure-treated hearts, bcl-2 was up-regulated (2.49 +/- 0.6 vs 1.16 +/- 0.11, controls, p < 0.005), whereas TNF-alpha was unchanged (396.1 +/- 100.38 vs 276.84 +/- 73.65 pg/mg, NS). Hearts treated with TGF-beta1 and pressure showed significant up-regulation of bcl-2 compared with hearts treated with TGF-beta1 without pressure (2.49 +/- 0.6 vs 1.17 +/- 0.6, p < 0.02). MPO showed no differences. Bcl-2 is down-regulated and TNF-alpha up-regulated in this model of ischemia-reperfusion injury. Furthermore, TGF-beta1 is linked to this process and ameliorates reperfusion injury by up-regulating bcl-2 and inhibiting TNF-alpha. Therapeutic overexpression of myocardial TGF-beta1 may be clinically useful to control ischemia-reperfusion injury associated with cardiac transplantation.

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.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

BCL2 oncogene translocation is mediated by a chi-like consensus

PubMed Central

1992-01-01

Examination of 64 translocations involving the major breakpoint region (mbr) of the BCL2 oncogene and the immunoglobulin heavy chain locus identified three short (14, 16, and 18 bp) segments within the mbr at which translocations occurred with very high frequency. Each of these clusters was associated with a 15-bp region of sequence homology, the principal one containing an octamer related to chi, the procaryotic activator of recombination. The presence of short deletions and N nucleotide additions at the breakpoints, as well as involvement of JH and DH coding regions, suggested that these sequences served as signals capable of interacting with the VDJ recombinase complex, even though no homology with the traditional heptamer/spacer/nonamer (IgRSS) existed. Furthermore, the BCL2 signal sequences were employed in a bidirectional fashion and could mediate recombination of one mbr region with another. Segments homologous to the BCL2 signal sequences flanked individual members of the XP family of diversity gene segments, which were themselves highly overrepresented in the reciprocal products (18q-) of BCL2 translocation. We propose that the chi-like signal sequences of BCL2 represent a distinct class of recognition sites for the recombinase complex, responsible for initiating interactions between regions of DNA separated by great distances, and that BCL2 translocation begins by a recombination event between mbr and DXP chi signals. Since recombinant joints containing chi, not IgRSS, occur in brain cells expressing RAG-1 (Matsuoka, M., F. Nagawa, K. Okazaki, L. Kingsbury, K. Yoshida, U. Muller, D. T. Larue, J. A. Winer, and H. Sakano. 1991. Science [Wash. DC]. 254:81; reference 1), we further suggest that the product of this gene could mediate both BCL2 translocation and the first step of normal DJ assembly through the creation of chi joints, rather than signal or coding joints. PMID:1588282

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

Effects of Lipoic Acid on Antiapoptotic Genes in Control and Ethanol-Treated Fetal Rhombencephalic Neurons

PubMed Central

Antonio, Angeline M.; Gillespie, Roberta A.; Druse, Mary J.

2011-01-01

This laboratory showed that ethanol augments apoptosis in fetal rhombencephalic neurons and co-treatment with alpha-lipoic acid (LA) or one of several other antioxidants prevents ethanol-associated apoptosis. Because ethanol increases oxidative stress, which causes apoptosis, it is likely that some of the neuroprotective effects of LA and other antioxidants involve classical antioxidant actions. Considering the reported link of LA with pro-survival cell signaling, it is also possible that LA’s neuroprotective effects involve additional mechanisms. The present study investigated the effects of LA on ethanol-treated fetal rhombencephalic neurons with regard to oxidative stress and up-regulation of the pro-survival genes Xiap and Bcl-2. We included parallel gene expression studies with N-acetyl cysteine (NAC) to determine whether LA’s effects on Xiap and Bcl-2 were shared by other antioxidants. We also used enzyme inhibitors to determine which signaling pathway(s) might be involved with the effects of LA. The results of this investigation showed that LA treatment of ethanol-treated neurons exerted several pro-survival effects. LA blocked two pro-apoptotic changes, i.e., the ethanol-associated rise in ROS and caspase-3. LA also up-regulated the expression genes that encode the anti-apoptotic proteins Bcl-2 and Xiap by a mechanism that involves NF-κB. NAC also up-regulated Bcl-2 and Xiap. Thus, the neuroprotective effects of LA and NAC could involve up-regulation of pro-survival genes as well as their classical antioxidant actions. PMID:21303669

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.

Double-hit lymphoma demonstrating t(6;14;18)(p25;q32;q21), suggesting two independent dual-hit translocations, MYC/BCL-2 and IRF4/BCL-2.

PubMed

Tabata, Rie; Yasumizu, Ryoji; Tabata, Chiharu; Kojima, Masaru

2013-01-01

Here, we report a rare case of double-hit lymphoma, demonstrating t(6;14;18)(p25;q32;q21), suggesting two independent dual-translocations, c-MYC/BCL-2 and IRF4/BCL-2. The present case had a rare abnormal chromosome, t(6;14;18)(p25;q32;q21), independently, in addition to known dual-hit chromosomal abnormalities, t(14;18)(q32;q21) and t(8;22)(q24;q11.2). Lymph node was characterized by a follicular and diffuse growth pattern with variously sized neoplastic follicles. The intrafollicular area was composed of centrocytes with a few centroblasts and the interfollicular area was occupied by uniformly spread medium- to large-sized lymphocytes. CD23 immunostaining demonstrated a disrupted follicular dendritic cell meshwork. The intrafollicular tumor cells had a germinal center phenotype with the expression of surface IgM, CD10, Bcl-2, Bcl-6, and MUM1/IRF4. However, the interfollicular larger cells showed plasmacytic differentiation with diminished CD20, Bcl-2, Bcl-6, and positive intracytoplasmic IgM, and co-expression of MUM1/IRF4 and CD138 with increased Ki-67-positive cells (> 90%). MUM1/IRF4 has been found to induce c-MYC expression, and in turn, MYC transactivates MUM1/IRF4, creating a positive autoregulatory feedback loop. On the other hand, MUM1/IRF4 functions as a tumor suppressor in c-MYC-induced B-cell leukemia. The present rare case arouses interest in view of the possible "dual" activation of both c-MYC and MUM1/IRF4 through two independent dual-translocations, c-MYC/BCL-2 and IRF4/BCL-2.

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

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

Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). Amore » super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.« less

Bcl-xL is overexpressed in hormone-resistant prostate cancer and promotes survival of LNCaP cells via interaction with proapoptotic Bak.

PubMed

Castilla, Carolina; Congregado, Belén; Chinchón, David; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

2006-10-01

Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.

Stress-induced activation of the brainstem Bcl-xL gene expression in rats treated with fluoxetine: correlations with serotonin metabolism and depressive-like behavior.

PubMed

Shishkina, Galina T; Kalinina, Tatyana S; Berezova, Inna V; Dygalo, Nikolay N

2012-01-01

Mechanisms underlying stress-induced depression and antidepressant drug action were shown to involve alterations in serotonergic (5-HT) neurotransmission and expression of genes coding for proteins associated with neurotrophic signaling pathways and cell-survival in the hippocampus and cortex. Expression of these genes in the brainstem containing 5-HT neurons may also be related to vulnerability or resilience to stress-related psychopathology. Here we investigated 5-HT markers and expression of genes for Brain-Derived Neurotrophic Factor (BDNF) and apoptotic proteins in the brainstem in relation to swim stress-induced behavioral despair. We found that anti-apoptotic Bcl-xL gene is sensitive to stress during the course of fluoxetine administration. Responsiveness of this gene to stress appeared concomitantly with an antidepressant-like effect of fluoxetine in the forced swim test. Bcl-xL transcript levels showed negative correlations with duration of immobility in the test and 5-HT turnover in the brainstem. In contrast, BDNF and pro-apoptotic protein Bax mRNA levels were unchanged by either fluoxetine or stress, suggesting specificity of Bcl-xL gene responses to these treatments. We also found that the levels of mRNAs for tryptophan hydroxylase-2 (TPH2) and 5-HT transporter (5-HTT) were significantly down-regulated following prolonged treatment with fluoxetine, but were not affected by stress. Unlike TPH2 and 5-HTT, 5-HT1A receptor mRNA levels were not altered by fluoxetine but significantly increased in response to swim stress. These data show that long-term fluoxetine treatment leads to changes in 5-HT and Bcl-xL responses to stress associated with antidepressant-like effects of the drug. This article is part of a Special Issue entitled 'Anxiety and Depression'. Copyright © 2011 Elsevier Ltd. All rights reserved.

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

PubMed Central

Faião-Flores, Fernanda; Coelho, Paulo Rogério Pinto; Toledo Arruda-Neto, João Dias; Maria-Engler, Silvya Stuchi; Tiago, Manoela; Capelozzi, Vera Luiza; Giorgi, Ricardo Rodrigues; Maria, Durvanei Augusto

2013-01-01

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT. PMID:23527236

Phospho-Bcl-x(L)(Ser62) plays a key role at DNA damage-induced G(2) checkpoint.

PubMed

Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2012-06-01

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G 2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G 2 arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G 2 arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G 2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G 2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G 2 arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.

Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells.

PubMed

Reshi, Latif; Wang, Hua-Ven; Hui, Cho-Fat; Su, Yu-Chin; Hong, Jiann-Ruey

2017-02-01

Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polymorphisms of the GR and HSD11B1 genes influence body mass index and weight gain during hormone replacement treatment in patients with Addison's disease.

PubMed

Molnár, Ágnes; Kövesdi, Annamária; Szücs, Nikolette; Tóth, Miklós; Igaz, Péter; Rácz, Károly; Patócs, Attila

2016-08-01

Glucocorticoid substitution is essential in patients with chronic primary adrenocortical insufficiency (Addison's disease) and both over-treatment and inadequate dosage have deleterious effects. Individual sensitivity to glucocorticoids is partly genetically determined. To test the hypothesis whether the well-characterized SNPs of the GR and HSD11B1 genes may modulate the individual sensitivity to exogenous glucocorticoids and may influence clinical and/or laboratory parameters and the glucocorticoid substitution dosage in patients with Addison's disease. 68 patients with primary adrenocortical insufficiency were involved. Clinical and laboratory data, as well as the dosage of the hormone replacement therapy were collected. Peripheral blood DNA was isolated, and the GR and HSD11B1 SNPs were examined using allele-specific PCR or Taqman assay on Real Time PCR. The allele frequency of the GR N363S polymorphism was higher in patients compared to the control group and the disease appeared significantly earlier in patients harbouring the GR A3669G compared to noncarriers. These patients had higher ACTH level measured at the time of diagnosis. Homozygous BclI carriers had higher body mass index (BMI) and lower total hydrocortisone equivalent supplementation dose needed than heterozygous or noncarriers. The BMI and weight gain during hormone replacement therapy were also higher in carriers of the HSD11B1 rs4844880 treated with glucocorticoids other than dexamethasone. The BclI polymorphism of the GR gene and the rs4844880 of the HSD11B1 gene may contribute to weight gain and may affect the individual need of glucocorticoid substitution dose in these patients. © 2016 John Wiley & Sons Ltd.

Nonsyndromic cleft lip with or without cleft palate: Evidence of linkage to BCL3 in 17 multigenerational families

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

Stein, J.; Hecht, T.; Stal, S.

1995-08-01

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was testedmore » under two models, autosomal dominant with reduced penetrance and affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities {ge}50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families. 39 refs., 8 figs., 4 tabs.« less

B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma.

PubMed

Snuderl, Matija; Kolman, Olga K; Chen, Yi-Bin; Hsu, Jessie J; Ackerman, Adam M; Dal Cin, Paola; Ferry, Judith A; Harris, Nancy Lee; Hasserjian, Robert P; Zukerberg, Lawrence R; Abramson, Jeremy S; Hochberg, Ephraim P; Lee, Hang; Lee, Alfred I; Toomey, Christiana E; Sohani, Aliyah R

2010-03-01

B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements, also known as "double-hit" lymphomas (DHL), are rare neoplasms characterized by highly aggressive clinical behavior, complex karyotypes, and a spectrum of pathologic features overlapping with Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and B-lymphoblastic lymphoma/leukemia (B-LBL). The clinical and pathologic spectrum of this rare entity, including comparison to other high-grade B-cell neoplasms, has not been well defined. We conducted a retrospective analysis of clinical and pathologic features of 20 cases of DHL seen at our institution during a 5-year period. In addition, we carried out case-control comparisons of DHL with BL and International Prognostic Index (IPI)-matched DLBCL. The 11 men and 9 women had a median age of 63.5 years (range 32 to 91). Six patients had a history of grade 1 to 2 follicular lymphoma; review of the prior biopsy specimens in 2 of 5 cases revealed blastoid morphology. Eighteen patients had Ann Arbor stage 3 or 4 disease and all had elevated serum lactate dehydrogenase (LDH) levels at presentation. Extranodal disease was present in 17/20 (85%), bone marrow involvement in 10/17 (59%) and central nervous system (CNS) disease in 5/11 (45%). Nineteen patients were treated with combination chemotherapy, of whom 18 received rituximab and 14 received CNS-directed therapy. Fourteen patients (70%) died within 8 months of diagnosis. Median overall survival in the DHL group (4.5 mo) was inferior to both BL (P=0.002) and IPI-matched DLBCL (P=0.04) control patients. Twelve DHL cases (60%) were classified as B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL, 7 cases (35%) as DLBCL, not otherwise specified, and 1 case as B-LBL. Distinguishing features from BL included expression of Bcl2 (P<0.0001), Mum1/IRF4 (P=0.006), Ki-67 <95% (P<0.0001), and absence of EBV-EBER (P=0.006). DHL commonly contained the t(8;22) rather than the t(8;14) seen in most BL controls (P=0.001), and exhibited a higher number of chromosomal aberrations (P=0.0009). DHL is a high-grade B-cell neoplasm with a poor prognosis, resistance to multiagent chemotherapy, and clinical and pathologic features distinct from other high-grade B-cell neoplasms. Familiarity with the morphologic and immunophenotypic spectrum of DHL is important in directing testing to detect concurrent IGH-BCL2 and MYC rearrangements when a karyotype is unavailable. The aggressive clinical behavior and combination of genetic abnormalities seen in these cases may warrant categorization as a separate entity in future classifications and call for novel therapeutic approaches.

Identifying candidate genes for 2p15p16.1 microdeletion syndrome using clinical, genomic, and functional analysis

PubMed Central

Bagheri, Hani; Badduke, Chansonette; Qiao, Ying; Colnaghi, Rita; Abramowicz, Iga; Alcantara, Diana; Dunham, Christopher; Wen, Jiadi; Wildin, Robert S.; Nowaczyk, Malgorzata J.M.; Eichmeyer, Jennifer; Lehman, Anna; Maranda, Bruno; Martell, Sally; Shan, Xianghong; Lewis, Suzanne M.E.; O’Driscoll, Mark; Gregory-Evans, Cheryl Y.

2016-01-01

The 2p15p16.1 microdeletion syndrome has a core phenotype consisting of intellectual disability, microcephaly, hypotonia, delayed growth, common craniofacial features, and digital anomalies. So far, more than 20 cases of 2p15p16.1 microdeletion syndrome have been reported in the literature; however, the size of the deletions and their breakpoints vary, making it difficult to identify the candidate genes. Recent reports pointed to 4 genes (XPO1, USP34, BCL11A, and REL) that were included, alone or in combination, in the smallest deletions causing the syndrome. Here, we describe 8 new patients with the 2p15p16.1 deletion and review all published cases to date. We demonstrate functional deficits for the above 4 candidate genes using patients’ lymphoblast cell lines (LCLs) and knockdown of their orthologs in zebrafish. All genes were dosage sensitive on the basis of reduced protein expression in LCLs. In addition, deletion of XPO1, a nuclear exporter, cosegregated with nuclear accumulation of one of its cargo molecules (rpS5) in patients’ LCLs. Other pathways associated with these genes (e.g., NF-κB and Wnt signaling as well as the DNA damage response) were not impaired in patients’ LCLs. Knockdown of xpo1a, rel, bcl11aa, and bcl11ab resulted in abnormal zebrafish embryonic development including microcephaly, dysmorphic body, hindered growth, and small fins as well as structural brain abnormalities. Our multifaceted analysis strongly implicates XPO1, REL, and BCL11A as candidate genes for 2p15p16.1 microdeletion syndrome. PMID:27699255

Transcriptional profiles of SHH pathway genes in keratocystic odontogenic tumor and ameloblastoma.

PubMed

Gurgel, Clarissa Araújo Silva; Buim, Marcilei Eliza Cavichiolli; Carvalho, Kátia Cândido; Sales, Caroline Brandi Schlaepfer; Reis, Mitermayer Galvão; de Souza, Renata Oliveira; de Faro Valverde, Ludmila; de Azevedo, Roberto Almeida; Dos Santos, Jean Nunes; Soares, Fernando Augusto; Ramos, Eduardo Antônio Gonçalves

2014-09-01

Sonic hedgehog (SHH) pathway activation has been identified as a key factor in the development of many types of tumors, including odontogenic tumors. Our study examined the expression of genes in the SHH pathway to characterize their roles in the pathogenesis of keratocystic odontogenic tumors (KOT) and ameloblastomas (AB). We quantified the expression of SHH, SMO, PTCH1, SUFU, GLI1, CCND1, and BCL2 genes by qPCR in a total of 23 KOT, 11 AB, and three non-neoplastic oral mucosa (NNM). We also measured the expression of proteins related to this pathway (CCND1 and BCL2) by immunohistochemistry. We observed overexpression of SMO, PTCH1, GLI1, and CCND1 genes in both KOT (23/23) and AB (11/11). However, we did not detect expression of the SHH gene in 21/23 KOT and 10/11 AB tumors. Low levels of the SUFU gene were expressed in KOT (P = 0.0199) and AB (P = 0.0127) relative to the NNM. Recurrent KOT exhibited high levels of SMO (P = 0.035), PTCH1 (P = 0.048), CCND1 (P = 0.048), and BCL2 (P = 0.045) transcripts. Using immunolabeling of CCND1, we observed no statistical difference between primary and recurrent KOT (P = 0.8815), sporadic and NBCCS-KOT (P = 0.7688), and unicystic and solid AB (P = 0.7521). Overexpression of upstream (PTCH1 and SMO) and downstream (GLI1, CCND1 and BCL2) genes in the SHH pathway leads to the constitutive activation of this pathway in KOT and AB and may suggest a mechanism for the development of these types of tumors. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hemoglobins emerging roles in mental disorders. Metabolical, genetical and immunological aspects.

PubMed

Altinoz, Meric A; Ince, Bahri

2017-10-01

Hemoglobin (Hb) expression in the central nervous system is recently shown. Cooccurences of mental disorders (mainly bipolar disorder (BD) and tic disorders) with β- or α-thalassemia trait or erythrocytosis were witnessed, which may be due to peripheral or central hypoxia/hyperoxia or haplotypal gene interactions. β-Globin genes reside at 11p15.5 close to tyrosine hydroxylase, dopamine receptor DRD4 and Brain Derived Neurotrophic Factor, which involve in psychiatric diseases. α-Globin genes reside at 16p13.3 which associates with BD, tic disorders, ATR-16 Syndrome and Rubinstein Taybi Syndrome (RTS). CREB-Binding Protein (CEBBP)-gene is mutated in RTS, which commonly associates with mood disorders. 16p13.3 region also contains GRIN2A gene encoding N-methyl-d-aspartate receptor-2A and SSTR5 (Somatostatin Receptor-5), again involving in mental disorders. We demonstrated a protective role of minor HbA2 against post-partum episodes in BD and association of higher minor HbF (fetal hemoglobin) levels with family history of psychosis in a BD-patient cohort. HbA2 increases in cardiac ischemia and in mountain dwellers indicating its likely protection against ischemia/hypoxia. HMGIY, a repressive transcription factor of δ-globin chain of HbA2 is increased in lymphocytes of schizophrenics. In autism, deletional mutations were found in BCL11A gene, which cause persistence of HbF at high levels in adulthood. Also, certain polymorphisms in BCL11A strongly associate with schizophrenia. Further, many drugs from anabolic steroids to antimalarial agents elevate HbF and may cause mania. We ascribe a protective role to HbA2 and a maladaptive detrimental role to HbF in psychopathology. We believe that future studies on hemoglobins may pave to discover novel pathogenesis mechanisms in mental disorders. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Zebrafish bcl2l is a survival factor in thyroid development.

PubMed

Porreca, Immacolata; De Felice, Elena; Fagman, Henrik; Di Lauro, Roberto; Sordino, Paolo

2012-06-15

Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Correlation Among Six Biologic Factors (p53, p21{sup WAF1}, MIB-1, EGFR, HER2, and Bcl-2) and Clinical Outcomes After Curative Chemoradiation Therapy in Squamous Cell Cervical Cancer

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

Yamashita, Hideomi; Murakami, Naoya; Asari, Takao

Purpose: The expressions of six cell-cycle-associated proteins were analyzed in cervical squamous cell carcinomas in correlation in a search for prognostic correlations in tumors treated with concurrent chemoradiation therapy (cCRT). Methods and Materials: The expressions of p53, p21/waf1/cip1, molecular immunology borstel-1 (MIB-1), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor type 2 (HER2), and Bcl-2 were studied using an immunohistochemical method in 57 cases of cervical squamous cell carcinoma treated with cCRT. Patients received cCRT between 1998 and 2005. The mean patient age was 61 years (range, 27-82 years). The number of patients with Stage II, III, andmore » IVA disease was 18, 29, and 10, respectively. Results: The number of patients with tumors positive for p53, p21/waf1/cip1, MIB-1, EGFR, HER2, and Bcl-2 was 26, 24, 49, 26, 13, and 11, respectively; no significant correlation was noted. The 5-year overall survival rates of HER2-positive and -negative patients was 76% vs. 44%, which was of borderline significance (p = 0.0675). No significant correlation was noted between overall survival and expressions of p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2. No correlation was observed between local control and expression of any of the proteins. Conclusion: Expression of HER2 protein had a weak impact of borderline significance on overall survival in squamous cell carcinoma of the uterine cervix treated with cCRT. However, no clinical associations could be established for p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2 protein expressions.« less

Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores

PubMed Central

Sinha, Rakesh K.; Pospíšil, Pavel; Maheshwari, Priti; Eudes, François

2016-01-01

Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity. PMID:28082995

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

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.

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

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

Heat-induced fibrillation of BclXL apoptotic repressor.

PubMed

Bhat, Vikas; Olenick, Max B; Schuchardt, Brett J; Mikles, David C; Deegan, Brian J; McDonald, Caleb B; Seldeen, Kenneth L; Kurouski, Dmitry; Faridi, Mohd Hafeez; Shareef, Mohammed M; Gupta, Vineet; Lednev, Igor K; Farooq, Amjad

2013-09-01

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease. Copyright © 2013 Elsevier B.V. All rights reserved.

CXCL12 mediates immunosuppression in the lymphoma microenvironment after allogeneic transplantation of hematopoietic cells.

PubMed

Dürr, Christoph; Pfeifer, Dietmar; Claus, Rainer; Schmitt-Graeff, Annette; Gerlach, Ulrike V; Graeser, Ralph; Krüger, Sophie; Gerbitz, Armin; Negrin, Robert S; Finke, Jürgen; Zeiser, Robert

2010-12-15

Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT. ©2010 AACR.

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

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.

N-Acetylcysteine Attenuates Ischemia-Reperfusion-Induced Apoptosis and Autophagy in Mouse Liver via Regulation of the ROS/JNK/Bcl-2 Pathway

PubMed Central

Xia, Yujing; Dai, Weiqi; Wang, Fan; Shen, Miao; Cheng, Ping; Wang, Junshan; Lu, Jie; Zhang, Yan; Yang, Jing; Zhu, Rong; Zhang, Huawei; Li, Jingjing; Zheng, Yuanyuan; Zhou, Yingqun; Guo, Chuanyong

2014-01-01

Background Hepatic ischemia–reperfusion injury (HIRI) remains a pivotal clinical problem after hemorrhagic shock, transplantation, and some types of toxic hepatic injury. Apoptosis and autophagy play important roles in cell death during HIRI. It is also known that N-acetylcysteine (NAC) has significant pharmacologic effects on HIRI including elimination of reactive oxygen species (ROS) and attenuation of hepatic apoptosis. However, the effects of NAC on HIRI-induced autophagy have not been reported. In this study, we evaluated the effects of NAC on autophagy and apoptosis in HIRI, and explored the possible mechanism involved. Methods A mouse model of segmental (70%) hepatic warm ischemia was adopted to determine hepatic injury. NAC (150 mg/kg), a hepatoprotection agent, was administered before surgery. We hypothesized that the mechanism of NAC may involve the ROS/JNK/Bcl-2 pathway. We evaluated the expression of JNK, P-JNK, Bcl-2, Beclin 1 and LC3 by western blotting and immunohistochemical staining. Autophagosomes were evaluated by transmission electron microscopy (TEM). Results We found that ALT, AST and pathological changes were significantly improved in the NAC group. Western blotting analysis showed that the expression levels of Beclin 1 and LC3 were significantly decreased in NAC-treated mice. In addition, JNK, p-JNK, Bax, TNF-α, NF-κB, IL2, IL6 and levels were also decreased in NAC-treated mice. Conclusion NAC can prevent HIRI-induced autophagy and apoptosis by influencing the JNK signal pathway. The mechanism is likely to involve attenuation of JNK and p-JNK via scavenged ROS, an indirect increase in Bcl-2 level, and finally an alteration in the balance of Beclin 1 and Bcl-2. PMID:25264893

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.

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

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 .

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

The human mitochondrial NADH: Ubiquinone oxidoreductase 51-kDa subunit oxidoreductase 51-kDa subunit maps adjacent to the glutathione S-transferase P1-1 gene on chromosome 11q13

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

Spencer, S.R.; Taylor, J.B.; Cowell, I.G.

The soluble glutathione transferases (GSTs) are a family of dimeric isoenymes catalyzing the conjugation of glutathione to hydrophobic electropiles. Their subunits can be grouped into four families, alpha, mu, pi, and theta, on the basis of their primary structures. In man, the pi class is represented by a single gene, GSTP1-1 (GST[pi]) localized to human chromosome 11, band q13. The oncogenes INT2, HSTF1, and PRAD1 are also localized at 11q13, and together with the GSTP1 locus and other gene loci mapped to 11q13, i.e., BCL1 and EMS1, they form a unit of DNA approximately 2000-2500 kb, known as the 11q13more » amplicon, which is often amplified in a range of solid tumors. Any gene locus at 11q13 is of interest because it may influence tumorigenesis. 14 refs., 1 fig.« less

Telomerase Is Involved in IL-7-Mediated Differential Survival of Naive and Memory CD4+ T Cells1

PubMed Central

Yang, Yinhua; An, Jie; Weng, Nan-ping

2008-01-01

IL-7 plays an essential role in T cell maintenance and survival. The survival effect of IL-7 is thought to be mediated through regulation of Bcl2 family proteins. After a comparative analysis of IL-7-induced growth and cell death of human naive and memory CD4+ T cells, we observed that more memory CD4+ T cells underwent cell division and proceeded to apoptosis than naive cells in response to IL-7. However, IL-7-induced expressions of Bcl2 family members (Bcl2, Bcl-xL, Bax, and Bad) were similar between naive and memory cells. Instead, we found that IL-7 induced higher levels of telomerase activity in naive cells than in memory cells, and the levels of IL-7-induced telomerase activity had a significant inverse correlation with cell death in CD4+ T cells. Furthermore, we showed that reducing expression of telomerase reverse transcriptase and telomerase activity significantly increased cell death of IL-7-cultured CD4+ T cells. Together, these findings demonstrate that telomerase is involved in IL-7-mediated differential survival of naive and memory CD4+ T cells. PMID:18322183

Methamphetamine induces apoptosis in immortalized neural cells: protection by the proto-oncogene, bcl-2.

PubMed

Cadet, J L; Ordonez, S V; Ordonez, J V

1997-02-01

Methamphetamine (METH) is an amphetamine analog that produces degeneration of the dopaminergic system in mammals. The neurotoxic effects of the drug are thought to be mediated by oxygen-based free radicals. In the present report, we have used immortalized neural cells obtained from rat mesencephalon in order to further assess the role of oxidative stress in METH-induced neurotoxicity. We thus tested if the anti-death proto-oncogene, bcl-2 could protect against METH-induced cytotoxicity. METH caused dose-dependent loss of cellular viability in control cells while bcl-2-expressing cells were protected against these deleterious effects. Using flow cytometry, immunofluorescent staining, and DNA electrophoresis, we also show that METH exposure can cause DNA strand breaks, chromatin condensation, nuclear fragmentation, and DNA laddering. All these changes were prevented by bcl-2 expression. These observations provide further support for the involvement of oxidative stress in the toxic effects of amphetamine analogs. They also document that METH-induced cytotoxicity is secondary to apoptosis. These findings may be of relevance to the cause(s) of Parkinson's disease which involves degeneration of the nigrostriatal dopaminergic pathway.

[Apoptosis and pathological process].

PubMed

Rami, Mukhammed Salim Iusef

2007-01-01

Apoptosis (programmed cell death) occurs normally for maitenance of tissue homeostasis and play an important role in morphogenesis, embriogenesis and tissue growth. On the other hand, apoptosis may be involved in different pathological processes such as malignancy, infectious diseases and autoimmune disorders. Apoptosis is regulated by various mediators. Caspases, death receptors, mitochondria, Bcl-2 protoncogenes and tumor supressor genes are considered to be the most important of them. Advance in apoptosis regulation research suggests enormouse facilities for therapy of wide range of human illnesses.

Targeting BCL2 With BH3 Mimetics: Basic Science and Clinical Application of Venetoclax in Chronic Lymphocytic Leukemia and Related B Cell Malignancies

PubMed Central

Huang, DCS

2016-01-01

The intracellular protein B‐cell‐lymphoma‐2 (BCL2) has been considered an attractive target for cancer therapy since the discovery of its function as a major promoter of cell survival (an anti‐apoptotic) in the late 1980s. However, the challenges of targeting a protein‐protein interaction delayed the discovery of fit‐for‐purpose molecules until the mid‐2000s. Since then, a series of high affinity small organic molecules that inhibits the interaction of BCL2 with the apoptotic machinery, the so‐called BH3‐mimetics, have been developed. Venetoclax (formerly ABT‐199) is the first to achieve US Food and Drug Administration approval, with an indication for treatment of patients with previously treated chronic lymphocytic leukemia (CLL) bearing deletion of the long arm of chromosome 17. Here, we review key aspects of the science underpinning the clinical application of BCL2 inhibitors and explore both our current knowledge and unresolved questions about its clinical utility, both in CLL and in other B‐cell malignancies that highly express BCL2. PMID:27806433

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.

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.

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.

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

Involvement of microRNA-133 and -29 in cardiac disturbances in diabetic ovariectomized rats.

PubMed

Habibi, Parisa; Alihemmati, Alireza; Nasirzadeh, Mohammadreza; Yousefi, Hadi; Habibi, Mohammadrasoul; Ahmadiasl, Nasser

2016-11-01

Menopause and diabetes obviously increase the risk of cardiovascular disease in women. The aims of the present study were to evaluate the effects of ovariectomy in type 2 diabetes on the histology and expression of miRNA-29, miRNA-133, IGF-1 and Bcl-2 genes and Bcl-2 protein and caspase 3 activity in the hearts of female rats. Forty Female Wistar rats were divided into four groups: control, sham, ovariectomized (OVX), and ovariectomized with type 2 diabetes (OVX.D). After the 8-week experiment, the histological evaluation of the heart tissue was performed using H&E staining and PAS analysis, and cardiac expression of miRNA-29, miRNA-133, IGF-1, and Bcl-2 were evaluated using real-time PCR, and Bcl-2 protein and caspase 3 activity were evaluated using Western blot and ELISA. Ovariectomy significantly decreased miRNA-29, miRNA-133, IGF-1, and BCL-2 expression and Bcl-2 protein and increased caspase 3 activity in the heart compared to sham animals group (P<0.05). Type 2 diabetes in ovariectomized rats markedly decreased expression of miRNA-29, miRNA-133, IGF-1, BCL-2 genes, and Bcl-2 protein, and increased caspase 3 activity and reduced collagen and fibroblast tissue and glycogen granule deposition in relation to OVX group (P<0.05). Our findings suggest that type 2 diabetes and menopause synergically could enhance the cardiac fibrosis through dysregulation of miRNA-29, miRNA-133, IGF-1, and Bcl-2 genes expression and Bcl-2 protein and upregulation of caspase 3 activity.

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

Cognitive analysis of schizophrenia risk genes that function as epigenetic regulators of gene expression.

PubMed

Whitton, Laura; Cosgrove, Donna; Clarkson, Christopher; Harold, Denise; Kendall, Kimberley; Richards, Alex; Mantripragada, Kiran; Owen, Michael J; O'Donovan, Michael C; Walters, James; Hartmann, Annette; Konte, Betina; Rujescu, Dan; Gill, Michael; Corvin, Aiden; Rea, Stephen; Donohoe, Gary; Morris, Derek W

2016-12-01

Epigenetic mechanisms are an important heritable and dynamic means of regulating various genomic functions, including gene expression, to orchestrate brain development, adult neurogenesis, and synaptic plasticity. These processes when perturbed are thought to contribute to schizophrenia pathophysiology. A core feature of schizophrenia is cognitive dysfunction. For genetic disorders where cognitive impairment is more severe such as intellectual disability, there are a disproportionally high number of genes involved in the epigenetic regulation of gene transcription. Evidence now supports some shared genetic aetiology between schizophrenia and intellectual disability. GWAS have identified 108 chromosomal regions associated with schizophrenia risk that span 350 genes. This study identified genes mapping to those loci that have epigenetic functions, and tested the risk alleles defining those loci for association with cognitive deficits. We developed a list of 350 genes with epigenetic functions and cross-referenced this with the GWAS loci. This identified eight candidate genes: BCL11B, CHD7, EP300, EPC2, GATAD2A, KDM3B, RERE, SATB2. Using a dataset of Irish psychosis cases and controls (n = 1235), the schizophrenia risk SNPs at these loci were tested for effects on IQ, working memory, episodic memory, and attention. Strongest associations were for rs6984242 with both measures of IQ (P = 0.001) and episodic memory (P = 0.007). We link rs6984242 to CHD7 via a long range eQTL. These associations were not replicated in independent samples. Our study highlights that a number of genes mapping to risk loci for schizophrenia may function as epigenetic regulators of gene expression but further studies are required to establish a role for these genes in cognition. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak

NASA Astrophysics Data System (ADS)

Zheng, Jing-Yi; Boustany, Nada N.

2010-07-01

Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 μM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 μm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

Immunohistochemical expression of protein 53, murine double minute 2, B-cell lymphoma 2, and proliferating cell nuclear antigen in odontogenic cysts and keratocystic odontogenic tumor.

PubMed

Galvão, Hebel Cavalcanti; Gordón-Núñez, Manuel Antonio; de Amorim, Rivadavio Fernandes Batista; Freitas, Roseana de Almeida; de Souza, Lelia Batista

2013-01-01

Even though odontogenic cysts share a similar histogenesis, they show different growth and differentiation profile due to differences in the proliferative cellular activity. We perform an immunohistochemical assessment of protein 53 (p53), proliferating cell nuclear antigen (PCNA), B-cell lymphoma 2 (bcl-2), and murine double minute 2 (MDM2) expression in odontogenic cysts and keratocystic odontogenic tumor analyzing their correlation with the biological behavior of these lesions. By the streptavidin-biotin-peroxidase method with antibodies against p53, PCNA, bcl-2, and MDM2 proteins, 11 radicular cysts, 11 dentigerous cysts, and 11 keratocystic odontogenic tumor were analyzed. The non-parametric Mann-Whitney U-test and Kruskall-Wallis test (P ≤ 0.05) were used to analyze the data. Immunopositivity for PCNA was observed in all cases appraised, predominantly in the suprabasal layer of keratocystic odontogenic tumor epithelial lining (SD ± 19.44), but no significant differences were found among the groups of lesions. Bcl-2 immunoexpression was observed especially in the basal layer of keratocystic odontogenic tumor. PCNA LI was significantly higher than bcl-2 LI in keratocystic odontogenic tumor. MDM2 and p53 immunoexpression were not detected in the lesions studied. Among the evaluated lesions, the keratocystic odontogenic tumor showed different immunoexpression of the proliferation and apoptosis markers. The results of this study suggest that the keratocystic odontogenic tumor presents distinct biological behavior of the odontogenic cysts, as for the processes of proliferation, apoptosis, and differentiation, reinforcing the information in favor of the neoplastic nature of this lesion.

No dramatic age-related loss of hair cells and spiral ganglion neurons in Bcl-2 over-expression mice or Bax null mice

PubMed Central

2010-01-01

Age-related decline of neuronal function is associated with age-related structural changes. In the central nervous system, age-related decline of cognitive performance is thought to be caused by synaptic loss instead of neuronal loss. However, in the cochlea, age-related loss of hair cells and spiral ganglion neurons (SGNs) is consistently observed in a variety of species, including humans. Since age-related loss of these cells is a major contributing factor to presbycusis, it is important to study possible molecular mechanisms underlying this age-related cell death. Previous studies suggested that apoptotic pathways were involved in age-related loss of hair cells and SGNs. In the present study, we examined the role of Bcl-2 gene in age-related hearing loss. In one transgenic mouse line over-expressing human Bcl-2, there were no significant differences between transgenic mice and wild type littermate controls in their hearing thresholds during aging. Histological analysis of the hair cells and SGNs showed no significant conservation of these cells in transgenic animals compared to the wild type controls during aging. These data suggest that Bcl-2 overexpression has no significant effect on age-related loss of hair cells and SGNs. We also found no delay of age-related hearing loss in mice lacking Bax gene. These findings suggest that age-related hearing loss is not through an apoptotic pathway involving key members of Bcl-2 family. PMID:20637089

Compromising σ-1 Receptors at the Endoplasmic Reticulum Render Cytotoxicity to Physiologically Relevant Concentrations of Dopamine in a Nuclear Factor-κB/Bcl-2-Dependent Mechanism: Potential Relevance to Parkinson's Disease

PubMed Central

Mori, Tomohisa; Hayashi, Teruo

2012-01-01

The endoplasmic reticulum (ER) chaperone σ-1 receptor (Sig-1R) is cytoprotective against ER stress-induced apoptosis. The level of Sig-1Rs in the brain was reported to be lower in early parkinsonian patients. Because dopamine (DA) toxicity is well known to be involved in the etiology of Parkinson's disease, we tested in this study whether a relationship might exist between Sig-1Rs and DA-induced cytotoxicity in a cellular model by using Chinese hamster ovary (CHO) cells. DA in physiological concentrations (e.g., lower than 10 μM) does not cause apoptosis. However, the same concentrations of DA cause apoptosis in Sig-1R knockdown CHO cells. In search of a mechanistic explanation, we found that unfolded protein response is not involved. Rather, the level of protective protein Bcl-2 is critically involved in this DA/Sig-1R knockdown-induced apoptosis. Specifically, the DA/Sig-1R knockdown causes a synergistic proteasomal conversion of nuclear factor κB (NF-κB) p105 to the active form of p50, which is known to down-regulate the transcription of Bcl-2. It is noteworthy that the DA/Sig-1R knockdown-induced apoptosis is blocked by the overexpression of Bcl-2. Our results therefore indicate that DA is involved in the activation of NF-κB and suggest that endogenous Sig-1Rs are tonically inhibiting the proteasomal conversion/activation of NF-κB caused by physiologically relevant concentrations of DA that would otherwise cause apoptosis. Thus, Sig-1Rs and associated ligands may represent new therapeutic targets for the treatment of parkinsonism. PMID:22399814

Neuroprotective effects of Bcl-2 overexpression on nerve cells of rats with acute cerebral infarction.

PubMed

Zhang, H R; Peng, J H; Zhu, G Y; Xu, R X

2015-07-13

We aimed to investigate the influence of lentiviral-mediated Bcl-2 overexpression in cerebral tissues of rats with acute cerebral infarction. Forty-five rats were randomly divided into sham, model, and treatment groups. The sham and model groups were administered a control lentiviral vector via the intracranial arteries 10 days before surgery, while the treatment group received lentivirus encoding a Bcl-2 overexpression vector. We induced cerebral artery infarction using a suture-occlusion method and analyzed the cerebral expression levels of apoptosis-related genes (caspase-3, Bax), total cerebral apoptosis, range of cerebral tissue infarction, and changes in nerve cell function after 72 h. The Bcl-2-encoding lentivirus was well expressed in rat cerebral tissues. The treatment group had significantly higher expression levels of Bcl-2 than the other two groups. After cerebral infarction, the model group had significantly increased expression levels of caspase-3 and Bax protein in cerebral tissues than the sham (P < 0.05). Expression of these apoptosis-related proteins in the treatment group was obviously lower than that in the model group (P < 0.05), but significantly higher than in the sham group (P < 0.05). Compared to sham, neuronal apoptosis levels and infarction range of cerebral tissues was increased in the model and treatment groups; however, these values in the treatment group were significantly lower than that in the model group (P < 0.05). Importantly, the treatment group had significantly decreased neurological impairment scores (P < 0.05). In conclusion, Bcl-2 over-expression can decrease neuronal apoptosis in rat cerebral tissue, and thus is neuroprotective after cerebral ischemia.

How I treat double-hit lymphoma.

PubMed

Friedberg, Jonathan W

2017-08-03

The 2016 revision of the World Health Organization (WHO) classification for lymphoma has included a new category of lymphoma, separate from diffuse large B-cell lymphoma, termed high-grade B-cell lymphoma with translocations involving myc and bcl-2 or bcl-6 . These lymphomas, which occur in <10% of cases of diffuse large B-cell lymphoma, have been referred to as double-hit lymphomas (or triple-hit lymphomas if all 3 rearrangements are present). It is important to differentiate these lymphomas from the larger group of double-expressor lymphomas, which have increased expression of MYC and BCL-2 and/or BCL-6 by immunohistochemistry, by using variable cutoff percentages to define positivity. Patients with double-hit lymphomas have a poor prognosis when treated with standard chemoimmunotherapy and have increased risk of central nervous system involvement and progression. Double-hit lymphomas may arise as a consequence of the transformation of the underlying indolent lymphoma. There are no published prospective trials in double-hit lymphoma, however retrospective studies strongly suggest that aggressive induction regimens may confer a superior outcome. In this article, I review my approach to the evaluation and treatment of double-hit lymphoma, with an eye toward future clinical trials incorporating rational targeted agents into the therapeutic armamentarium. © 2017 by The American Society of Hematology.

SOX7 Suppresses Wnt Signaling by Disrupting β-Catenin/BCL9 Interaction.

PubMed

Fan, Rong; He, HaiYan; Yao, Wang; Zhu, YanFeng; Zhou, XunJie; Gui, MingTai; Lu, Jing; Xi, Hao; Deng, ZhongLong; Fan, Min

2018-02-01

The Wnt signaling is involved in angiogenesis and tumor development. β-catenin is the core component of the Wnt pathway, which mediates oncogenic transcription and regulated by a series of proteins. Sex-determining region Y-box 7 (SOX7) is a member of high-mobility-group transcription factor family, which inhibits oncogenic Wnt signaling in lots of tumor cells with unknown mechanism. By coimmunoprecipitation (co-IP) and super Topflash reporter assay, SOX7 can bind β-catenin and inhibit β-catenin/T cell factor (TCF)-mediated transcription. Meanwhile, B cell lymphoma 9 (BCL9) drives Wnt signaling path through direct binding-mediated β-catenin. Finally, we found that SOX7 inhibits oncogenic β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. Mechanistically, SOX7 compete with BCL9 to bind β-catenin. Our results show SOX7 inhibited Wnt signaling as suppressor and could be an important target for anticancer therapy.

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

PubMed

Matsumoto, Y; Kawatani, M; Simizu, S; Tanaka, T; Takada, M; Imoto, M

2000-01-01

The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

Lysosome-mediated Cell Death and Autophagy-Dependent Multidrug Resistance in Breast Cancer

DTIC Science & Technology

2008-10-01

gene links mitochondria and cell death, the data suggests that Bcl2 may be involved in autophagic cell death and AD-MDR. GeneGo analysis also...GSK3 beta GSK3 beta E2A p53 p21 p21 E2F1 PPAR -gamma JNK1(MA PK8) JNK1(M APK8) ESR1 (nuclear) RARalpha Androgen receptor Androge n receptor p53...RelA (p65 NF-kB subunit) Erk (MAPK1/3 ) Erk (MAPK1/ 3) PPAR - gamma SOX9 Bcl-2 Bcl-2 RARalpha SP1 EGFR EGFR RelA (p65 NF- kB subunit) RARalpha RelA

Effects of the bile acid UDCA on PDT efficacy in vitro and in vivo

NASA Astrophysics Data System (ADS)

Kessel, David; Castelli, Michelle; Sykes, Elizabeth; Garbo, Greta M.

2004-06-01

The phototoxicity of PDT in cell culture can be promoted by the relatively hydrophilic bile acid UDCA (ursodeoxycholic acid). This was attributed to a conformational change in the anti-apoptotic protein Bcl-2, leading to an enhanced sensitivity to photodamage by sensitizers that target sites of Bcl-2 localization. UDCA also promoted the binding and inactivation of Bcl-2 by the non-peptidic antagonist HA14- 1, suggesting that UDCA may also be useful for promoting chemotherapy designed to target Bcl-2. In tumor-bearing animals, addition of UDCA to a PDT protocol involving the tin etiopurpurin SnET2 resulted in enhanced cancer control, but there was no effect on the extent of PDT-induced vascular shut-down. These results are consistent with the propo proposal that UDCA only promotes direct tumor cell kill. In this report, we have sal summarized recent research relating to mode of action of UDCA as it effects the on the efficacy of photodynamic therapy where Bcl-2 is among the PDT targets, and discuss the implications of the results.

Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3.

PubMed

Sun, Yi; Fu, Amina; Xu, Wu; Chao, Jyh-Rong; Moshiach, Simon; Morris, Stephan W

2015-12-01

Myeloid leukemia factor 1 (MLF1) was involved in t(3;5) chromosomal rearrangement and aberrantly expressed in myelodysplastic syndromes/acute myeloid leukemia patients. Ex vivo experiments showed that the lymphocytes from the Mlf1-deficient mice were more resistant to apoptotic stimulations than the wild-type cells. Furthermore, the ectopically expressed MLF1 induced apoptosis in the cell models. These findings revealed that MLF1 was required for the cells to respond to the apoptotic stimulations. Ex vivo experiments also demonstrated that cytokine withdrawal significantly up-regulated Mlf1's expression and promoted its association with B cell lymphoma-extra large (Bcl-XL) in the lymphocytes, at the same time reduced the association of Bax with Bcl-XL The same effects were also observed in the cells that over-expressed MLF1. However, these effects were observed in Mlf1 null lymphocytes as well as the cells over-expressing Bcl-XL. In addition, MLF1's proapoptosis could be completely prevented by co-expression of Bcl-XL and significantly attenuated in Bax/Bak double null cells. These data, taken together, strongly suggested that in response to the stresses, up-regulated Mlf1 promoted its association with Bcl-XL and reduced the available Bcl-XL for associating with Bax, which resulted in releasing Bax from the Bcl-XL and apoptosis in turn. Lastly, we showed that MLF1 was negatively regulated by 14-3-3 and revealed that 14-3-3 bound to MLF1 and physically blocked MLF1's Bcl-2 homology domain 3 (BH3) as well as Bcl-XL from associating with MLF1. Our findings suggested that ectopically expressed MLF1 could be responsible for the pathological apoptosis in early myelodysplastic syndrome (MDS) patients.

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

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.

Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

PubMed

Karim, Zubair A; Hensch, Nicole R; Qasim, Hanan; Alshbool, Fatima Z; Khasawneh, Fadi T

2018-06-02

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitative Expression of C-Type Lectin Receptors in Humans and Mice

PubMed Central

Lech, Maciej; Susanti, Heni Eka; Römmele, Christoph; Gröbmayr, Regina; Günthner, Roman; Anders, Hans-Joachim

2012-01-01

C-type lectin receptors and their adaptor molecules are involved in the recognition of glycosylated self-antigens and pathogens. However, little is known about the species- and organ-specific expression profiles of these molecules. We therefore determined the mRNA expression levels of Dectin-1, MR1, MR2, DC-SIGN, Syk, Card-9, Bcl-10, Malt-1, Src, Dec-205, Galectin-1, Tim-3, Trem-1, and DAP-12 in 11 solid organs of human and mice. Mouse organs revealed lower mRNA levels of most molecules compared to spleen. However, Dec-205 and Galectin-1 in thymus, Src in brain, MR2, Card-9, Bcl-10, Src, and Dec-205 in small intestine, MR2, Bcl-10, Src, Galectin-1 in kidney, and Src and Galectin-1 in muscle were at least 2-fold higher expressed compared to spleen. Human lung, liver and heart expressed higher mRNA levels of most genes compared to spleen. Dectin-1, MR1, Syk and Trem-1 mRNA were strongly up-regulated upon ischemia-reperfusion injury in murine kidney. Tim3, DAP-12, Card-9, DC-SIGN and MR2 were further up-regulated during renal fibrosis. Murine kidney showed higher DAP-12, Syk, Card-9 and Dectin-1 mRNA expression during the progression of lupus nephritis. Thus, the organ-, and species-specific expression of C-type lectin receptors is different between mice and humans which must be considered in the interpretation of related studies. PMID:22949850

Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Taverna, Pietro; Cortes, Jorge E; Gandhi, Varsha

2011-07-21

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound, SGI-1776 inhibits Pim-1, Pim-2 and Pim-3, and was evaluated in AML-cell line, -xenograft model, and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets, c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47), were both decreased in actively cycling AML cell lines MV-4-11, MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2, Bcl-x(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data, xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly, SGI-1776 was also cytotoxic in AML primary cells, irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment.

Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Taverna, Pietro; Cortes, Jorge E.

2011-01-01

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound, SGI-1776 inhibits Pim-1, Pim-2 and Pim-3, and was evaluated in AML-cell line, -xenograft model, and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets, c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47), were both decreased in actively cycling AML cell lines MV-4-11, MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2, Bcl-xL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data, xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly, SGI-1776 was also cytotoxic in AML primary cells, irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. PMID:21628411

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.

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2.

PubMed

Long, Bo; Li, Na; Xu, Xi-Xia; Li, Xiao-Xin; Xu, Xin-Jie; Guo, Dan; Zhang, Dong; Wu, Zhi-Hong; Zhang, Shu-Yang

2018-01-01

Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

A de-novo interstitial microduplication involving 2p16.1-p15 and mirroring 2p16.1-p15 microdeletion syndrome: Clinical and molecular analysis.

PubMed

Mimouni-Bloch, Aviva; Yeshaya, Josepha; Kahana, Sarit; Maya, Idit; Basel-Vanagaite, Lina

2015-11-01

Microdeletions of various sizes in the 2p16.1-p15 chromosomal region have been grouped together under the 2p16.1-p15 microdeletion syndrome. Children with this syndrome generally share certain features including microcephaly, developmental delay, facial dysmorphism, urogenital and skeletal abnormalities. We present a child with a de-novo interstitial 1665 kb duplication of 2p16.1-p15. Clinical features of this child are distinct from those of children with the 2p16.1-p15 microdeletion syndrome, specifically the head circumference which is within the normal range and mild intellectual disability with absence of autistic behaviors. Microduplications many times bear milder clinical phenotypes in comparison with corresponding microdeletion syndromes. Indeed, as compared to the microdeletion syndrome patients, the 2p16.1-p15 microduplication seems to have a milder cognitive effect and no effect on other body systems. Limited information available in genetic databases about cases with overlapping duplications indicates that they all have abnormal developmental phenotypes. The involvement of genes in this location including BCL11A, USP34 and PEX13, affecting fundamental developmental processes both within and outside the nervous system may explain the clinical features of the individual described in this report. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

PubMed

Chiang, Wei-Chung; Wei, Yongjie; Kuo, Yi-Chun; Wei, Shuguang; Zhou, Anwu; Zou, Zhongju; Yehl, Jenna; Ranaghan, Matthew J; Skepner, Adam; Bittker, Joshua A; Perez, Jose R; Posner, Bruce A; Levine, Beth

2018-06-21

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC 50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

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 proteins, including Bcl-2, Bcl-xL, and p-AKT, in animal models of glaucoma. PMID:27587945

In situ detection of inflammatory cytokines and apoptosis in pemphigus foliaceus patients.

PubMed

Rodrigues, Denise Bertulucci Rocha; Pereira, Sanivia Aparecida Lima; dos Reis, Marlene Antônia; Adad, Sheila Jorge; Caixeta, João Eduardo; Chiba, Angélica Maeda; Sousa, Richard Atila; Rodrigues, Virmondes

2009-01-01

Endemic pemphigus foliaceus, or fogo selvagem, is a chronic autoimmune disease characterized by the formation of intraepidermal blisters that reduce adhesion between keratinocytes. Endemic pemphigus foliaceus is associated with the presence of autoantibodies and high levels of cytokines involved in the inflammatory response. To evaluate the expression of the proinflammatory cytokines interleukin 1, interferon gamma, and tumor necrosis factor alpha; the proapoptotic inducers Fas and inducible nitric oxide synthase; and the apoptosis inhibitor Bcl-2; and to evaluate the presence of apoptosis. Skin biopsies from 13 patients with endemic pemphigus foliaceus and controls were evaluated by immunohistochemistry and apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Proinflammatory cytokines were only detected in cells of the inflammatory exudate. Inducible nitric oxide synthase, Fas, and Bcl-2 were expressed by both epithelial and inflammatory cells. Epithelial apoptosis was observed in 12 cases (92.3%), and subepithelial apoptosis in 11 cases (85%). This study suggests that apoptosis as well as the local production of proinflammatory cytokines are associated with endemic pemphigus foliaceus lesions. These results may contribute to the development of new therapeutic approaches to endemic pemphigus foliaceus.

Immunohistochemical and prognostic analysis of apoptosis and proliferation in uveal melanoma.

PubMed Central

Mooy, C. M.; Luyten, G. P.; de Jong, P. T.; Luider, T. M.; Stijnen, T.; van de Ham, F.; van Vroonhoven, C. C.; Bosman, F. T.

1995-01-01

Neoplasia can be defined as deregulated tissue homeostasis caused by an imbalance between proliferation and apoptosis. Many genes are involved in the maintenance of tissue homeostasis, eg, the c-myc oncoprotein, which is an important regulator of cell proliferation and Bcl-2 protein, which is involved in the regulation of apoptosis. We studied retrospectively indices of proliferation, such as mitotic count and the Mib-1 index, on 51 uveal melanomas and compared their prognostic significance with established indicators of prognosis such as cell type and tumor size. Along the same line we investigated the expression of the regulating proteins c-myc and Bcl-2. Of all parameters tested, the largest tumor diameter and mitotic count were most strongly associated with tumor-related death (P < 0.001 and P = 0.005, respectively). In addition, cell type, the presence of epithelioid cells, the Mib-1 index, and the percentage of cytoplasmic c-myc-positive cells were significant predictive factors. Multivariate analysis showed that the Mib-1 index, largest tumor diameter, and the percentage of cytoplasmic c-myc-positive cells were independent prognostic parameters. Bcl-2 expression did not correlate with clinical outcome. The Mib-1 index correlated with the presence of epithelioid cells (P < 0.03) and the presence of apoptotic bodies (P < 0.001) and c-myc. A strong inverse relationship was found between (nuclear and cytoplasmic) c-myc and Bcl-2 (P < 0.00004 and P < 0.006, respectively), suggesting that Bcl-2 cooperates with c-myc to immortalize uveal melanoma cells. Images Figure 1 Figure 2 PMID:7573354

Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

PubMed Central

Ueda, Norishi

2015-01-01

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

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

Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation.

PubMed

Rouble, Andrew N; Hefler, Joshua; Mamady, Hapsatou; Storey, Kenneth B; Tessier, Shannon N

2013-01-01

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation

PubMed Central

Mamady, Hapsatou; Tessier, Shannon N.

2013-01-01

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor. PMID:23638364

Oncogenic HER2Δ16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors

PubMed Central

Cittelly, Diana M.; Das, Partha M.; Salvo, Virgilio A.; Fonseca, Juan P.; Burow, Matthew E.; Jones, Frank E.

2010-01-01

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis. PMID:20876285

Extranodal involvement of diffuse large B-cell lymphoma in the head and neck: An indicator of good prognosis.

PubMed

Lee, Doh Young; Kang, Karam; Jung, Hwaejoon; Park, Young Min; Cho, Jae-Gu; Baek, Seung-Kuk; Kwon, Soon-Young; Jung, Kwang-Yoon; Woo, Jeong-Soo

2018-05-31

In this study, we analyzed clinicopathological characteristics and survival outcomes according to extranodal involvement of diffuse large B-cell lymphoma (DLBCL) in the head and neck. A retrospective analysis was conducted on 110 patients from 2004 to 2014 with CD20-positive DLBCL involving the head and neck area. Patients were categorized into two groups, extranodal and nodal, according to involvement of extranodal sites in the head and neck. Outcome measurements for the groups included clinical response to treatment and recurrence rates. Palatine tonsils were the most frequently involved extranodal site in the head and neck (29.1%). Among clinicopathological parameters, proportion of patients with lactate dehydrogenase over 350 IU/L (p=0.033), cell of origin (p<0.001), and treatment outcomes (p=0.007) were significantly different between the two groups. Among cell origin markers CD10, Bcl6, and MUM1, MUM1 was significantly correlated with extranodal involvement (p=0.029). Recurrence rates were similar between groups, while disease-specific survival was significantly higher in the extranodal group (p=0.011). Disease-specific survival of the extranodal group was also higher than the nodal group with extranodal involvement of other body sites (p=0.010). Among patients with negative expression of CD10 (p=0.015), Bcl6 (p=0.018), and MUM1 (p=0.005), survival was longer in the extranodal than the nodal group. DLBCL patients with extranodal involvement of the head and neck may have longer survival outcomes than patients with solely nodal involvement. Increased survival may be more prominent in patients with negative expression of CD10, Bcl6, and MUM1. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Interactions Between Vitamin D and Breast Cancer

DTIC Science & Technology

2009-09-01

ERBB2 GRB7 B EGFR MKI67 AURKA BIRC5 CCND1 CCNA1 TP53 MMP11 CTSL2 BAX BCL2 VDR C CYP24A1 CYP27B1 HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF D...BIRC5 CCND1 CCNA1 TP53 MMP11 CTSL2 BAX BCL2 VDR G CYP24A1 CYP27B1 HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF H CDKN1A IGFBP3 SPP1 AR PTHLH...NAD) C08 Hs.199248 NM_000958 PTGER4 Prostaglandin E receptor 4 (subtype EP4) C09 Hs.497822 NM_007207 DUSP10 Dual specificity phosphatase 10 C10

CC genotype of anti-apoptotic gene BCL-2 (-938 C/A) is an independent prognostic marker of unfavorable clinical outcome in patients with non-small-cell lung cancer.

PubMed

Javid, J; Mir, R; Mirza, M; Imtiyaz, A; Prasant, Y; Mariyam, Z; Julka, P K; Mohan, A; Lone, M; Ray, P C; Saxena, A

2015-04-01

B cell lymphoma 2 (BCL-2) gene is a well-known regulator of apoptosis and a key element in cancer development and progression. A regulatory (-938C>A, rs2279115) single-nucleotide polymorphism in the inhibitory P2 BCL-2 gene promoter generates significantly different BCL-2 promoter activities and has been associated with different clinical outcomes in various malignancies. The aim of the present study was to analyze the possible influence of the (-938C>A) SNP on the risk and survival of Indian patients suffering from NSCLC. A hospital-based case-control study of 155 age- and sex-matched patients diagnosed with NSCLC and 155 cancer-free controls was conducted and genotyped by performing PIRA-PCR to elucidate the putative association between clinical outcome and genotypes of BCL-2 (-938C>A, rs2279115). The association of the polymorphism with the survival of NSCLC patients was analyzed by Kaplan-Meier curves. In Indian NSCLC, patients increased risk of developing NSCLC was found to be associated with BCL-2 (-938) CC genotype, [OR 3.68 (1.92-6.79), RR 1.87 (1.35-2.57) and RD 31.03 (16.79-45.27) p 0.00006 for CC and OR 2.08 (1.18-3.66), RR 1.36 (1.08-1.71) and RD 17.74 (4.68-30.81) p 0.01 for AC genotype]. Patients homozygous for C allele exhibited a significant poor overall survival compared with patients displaying AC + CC or AC or AA genotype [median survival (months) 8 vs. 11 vs. 14 vs. 35.5 (p < 0.0001)]. In addition, significant associations were observed between TNM stage, histological type, distant metastases status, family history of any cancer, gender and age group of NSCLC patients with BCL-2 (-938C>A) polymorphism. Genetic polymorphism in the inhibitory P2 promoter region of anti-apoptotic BCL-2 genes contributes to the risk of developing non-small-cell lung cancer in Indian population. BCL-2 (-938CC) genotype was an independent adverse prognostic factor for patients with NSCLC.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

PubMed

Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

2015-07-31

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells

PubMed Central

Lagadinou, Eleni D.; Sach, Alexander; Callahan, Kevin; Rossi, Randall M.; Neering, Sarah J.; Minhajuddin, Mohammad; Ashton, John M.; Pei, Shanshan; Grose, Valerie; O’Dwyer, Kristen M.; Liesveld, Jane L.; Brookes, Paul S.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

Summary Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally-defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly over-express BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation. PMID:23333149

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

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.

Expression of NF-kappaB dependent genes in human cells in response to heavy ion beams

NASA Astrophysics Data System (ADS)

Hellweg, Christine; Baumstark-Khan, Christa; Ruland, Rebecca; Schmitz, Claudia; Lau, Patrick; Testard, Isabelle; Reitz, Guenther

Space radiation is a primary concern for manned spaceflight and is a potentially limiting factor for long term orbital and interplanetary missions. Understanding of the cellular and molecular processes underlying cell death and transformation related events by space radiation may allow better risk estimation and development of appropriate countermeasures. The pathway leading to activation of the transcription factor nuclear factor κB (NF-κB) and increased transcription of its target gene might modulate cellular radiation response. Previous studies suggest a linear energy transfer (LET) dependency of transcription factor nuclear factor κB (NF-κB) activation: high LET radiation activates NF-κB more efficiently than low LET radiation. In this work, the relative expressions of several NF-κB regulated genes (Gadd45β, NFKBIA encoding the NF-κB inhibitor IκBα, and the anti-apoptotic genes XIAP, bcl-2, and bcl-xL) were examined by quantitative real-time Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Human embryonic cells with neuronal differentiation potential (HEK/293) were exposed to accelerated heavy ions or to X-rays (200 kV) or incubated in presence of the strong NF-κB activator tumor necrosis factor α (TNF-α). Target gene expression data were normalized to the expression index of several unregulated reference genes (B2M, GAPDH, PBGD, HPRT). NFKBIA expression is enhanced for 24 h after TNF-α treatment, while Gadd45β expression was only temporarily up-regulated. High doses of X-rays (8 and 16 Gy) and of 13 C ions (75 MeV/n, LET 33 keV/µm, 4.7 Gy) up-regulate NFKBIA and Gadd45β expression temporarily. 13 C ion with higher LET (35 MeV/n, 73 keV/µm) enhance NFKBIA expression already after 1 Gy, and a passing up-regulation of Bcl-2, bcl-xL and XIAP expression was observed 2 h after 0.5 Gy. 20 Ne (95 MeV/A, 80 keV/µm) and 36 Ar ions (95 MeV/A, 271 keV/µm) were the strongest inducers of Gadd45β, NFKBIA, and XIAP with doses from 0.5 to 3.8 Gy. 58 Ni (75 MeV/A, 906 keV/µm) and 208 Pb ion exposure (29 MeV/A, 9764 keV/µm) reduced the expression of Gadd45β. NFKBIA expression was enhanced after 58 Ni ion exposure, but down-regulated after 208 Pb ion exposure. Bcl-2 and Bcl-xL were mostly unaffected by the tested irradiation conditions or only transitorily up-regulated. In conclusion, genes involved in cell cycle regulation (Gadd45β), in inhibition of apoptosis (XIAP, bcl-2, and bcl-xL) and in control of the NF-κB pathway (NFKBIA) show a differentiated expression profile after exposure of human cells to heavy ions of different LET. This might be a step towards understanding of the previously observed LET dependency of cell survival and cell cycle arrest.

Defective expression of apoptosis-related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation.

PubMed

de Oliveira, G L V; Ferreira, A F; Gasparotto, E P L; Kashima, S; Covas, D T; Guerreiro, C T; Brum, D G; Barreira, A A; Voltarelli, J C; Simões, B P; Oliveira, M C; de Castro, F A; Malmegrim, K C R

2017-03-01

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis-related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)-based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIM EL , FAS, FASL, A1, BCL2, BCLX L , CFLIP L and CIAP2 genes were up-regulated after AHSCT. With the exception of BIK, BIM EL and A1, all genes reached levels similar to controls at day + 720 post-transplantation. Furthermore, in these patients, we observed increased CD8 + Fas + T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIP L and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day + 720 post-AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLX L and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl-2 expression before transplantation. At 1 year post-AHSCT, expression of Bak, Bim, Bcl-2, Bcl-xL and cFlip-L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis-related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re-establishment of immune tolerance during the first 2 years post-transplantation. © 2016 British Society for Immunology.

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.

Fumagillin treatment of hepatocellular carcinoma in rats: An in vivo study of antiangiogenesis

PubMed Central

Sheen, I-Shyan; Jeng, Kuo-Shyang; Jeng, Wen-Juei; Jeng, Chi-Juei; Wang, Yi-Ching; Gu, Shu-Ling; Tseng, Shin-Yun; Chu, Chien-Ming; Lin, Chia-Hui; Chang, Kuo-Ming

2005-01-01

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg·d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range 2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity. PMID:15682466

Nimbolide targets BCL2 and induces apoptosis in preclinical models of Waldenströms macroglobulinemia

PubMed Central

Chitta, K; Paulus, A; Caulfield, T R; Akhtar, S; Blake, M-KK; Ailawadhi, S; Knight, J; Heckman, M G; Pinkerton, A; Chanan-Khan, A

2014-01-01

Neem leaf extract (NLE) has medicinal properties, which have been attributed to its limonoid content. We identified the NLE tetranorterpenoid, nimbolide, as being the key limonoid responsible for the cytotoxicity of NLE in various preclinical models of human B-lymphocyte cancer. Of the models tested, Waldenströms macroglobulinemia (WM) cells were most sensitive to nimbolide, undergoing significant mitochondrial mediated apoptosis. Notably, nimbolide toxicity was also observed in drug-resistant (bortezomib or ibrutinib) WM cells. To identify putative targets of nimbolide, relevant in WM, we used chemoinformatics-based approaches comprised of virtual in silico screening, molecular modeling and target–ligand reverse docking. In silico analysis revealed the antiapoptotic protein BCL2 was the preferential binding partner of nimbolide. The significance of this finding was further tested in vitro in RS4;11 (BCL2-dependent) tumor cells, in which nimbolide induced significantly more apoptosis compared with BCL2 mutated (Jurkat BCL2Ser70-Ala) cells. Lastly, intraperitoneal administration of nimbolide in WM tumor xenografted mice, significantly reduced tumor growth and IgM secretion in vivo, while modulating the expression of several proteins as seen on immunohistochemistry. Overall, our data demonstrate that nimbolide is highly active in WM cells, as well as other B-cell cancers, and engages BCL2 to exert its cytotoxic activity. PMID:25382610

Annexin A11 knockdown inhibits in vitro proliferation and enhances survival of Hca-F cell via Akt2/FoxO1 pathway and MMP-9 expression.

PubMed

Liu, Shuqing; Wang, Jiasheng; Guo, Chunmei; Qi, Houbao; Sun, Ming-Zhong

2015-03-01

Annexin A11 (Anxa11), a Ca(2+)-regulated phospholipid-binding protein, is involved in cell apoptosis, differentiation, vesicle trafficking, cancer progression and autoimmune diseases. Previous study from our group indicated that Anxa11 was associated with lymphatic metastatic potential of murine hepatocarcinoma cells. Herein, we investigated the effects and action mechanism of Anxa11 knockdown on in vitro cell proliferation and apoptosis of Hca-F, a murine hepatocarcinoma cell with∼75% lymph node metastatic potential. Real-time PCR and western blotting assays indicated that Anxa11 was significantly downregulated in monoclonal Anxa11-shRNA-transfected Hca-F cells. Anxa11 knockdown in Hca-F suppressed its in vitro proliferation and cell apoptosis capacities. Following Anxa11 knockdown in Hca-F cells, Bax/Bcl-2 expression level ratio, Akt2 and FoxO1 (pSer319) expression levels as well as MMP-9 mRNA and active MMP-9 protein levels were significantly elevated in Hca-F cells. In conclusion, Annexin A11 knockdown inhibits the in vitro proliferation and cell apoptosis of Hca-F cell via Akt2/FoxO1 and/or MMP-9 expression pathway. Anxa11 might play an important role in hepatocarcinoma cell invasion and metastasis and hepatocarcinoma malignancy. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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 prognosis was found in the cases with low microvessel density (MVD) in tumor tissue and high expression of SPARC (SPARC≥5%) in stromal cells. "Double-hit" lymphoma was rare among primary gastric lymphoma, while patients with multiple gene amplification and/or copy gains of c-Myc, bcl2 and bcl6, and "double-protein" gastric B-cell lymphomas had a poor clinical outcome. In addition, patients with MYC, BCL2 and BCL6 expression negative or low MVD in tumor tissue with high expression of SPARC in stromal cells could have better prognosis than other gastric B-cell lymphomas regardless of their clinical stage and pathological types. These results would be of very importance for clinical stratified management and precision medicine of gastric B-cell lymphomas.

DNA methylation pattern of apoptosis-related genes in ameloblastoma.

PubMed

Costa, Sfs; Pereira, N B; Pereira, Kma; Campos, K; de Castro, W H; Diniz, M G; Gomes, C C; Gomez, R S

2017-09-01

DNA methylation is an important mechanism of gene control expression, and it has been poorly addressed in odontogenic tumours. On this basis, we aimed to assess the methylation pattern of 22 apoptosis-related genes in solid ameloblastomas. Ameloblastoma fresh samples (n = 10) and dental follicles (n = 8) were included in the study. The percentage fraction of methylated and unmethylated DNA promoter of 22 apoptosis-related genes was determined using enzymatic restriction digestion and quantitative real-time PCR (qPCR) array. The relative expressions of the genes that showed the most discrepant methylation profile between tumours and controls were analysed by reverse-transcription quantitative PCR (RT-qPCR). Lower methylation percentages of TNFRSF25 (47.2%) and BCL2L11 (33.2%) were observed in ameloblastomas compared with dental follicles (79.3% and 59.5%, respectively). The RT-qPCR analysis showed increased expression of BCL2L11 in ameloblastomas compared with dental follicles, in agreement with the methylation analysis results, while there was no difference between the expression levels of TNFRSF25 between both groups. On the basis of our results, the transcription of the apoptosis-related gene BCL2L11 is possibly regulated by promoter DNA methylation in ameloblastoma. The biological significance of this finding in ameloblastoma pathobiology remains to be clarified. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Targeting BCL2 With BH3 Mimetics: Basic Science and Clinical Application of Venetoclax in Chronic Lymphocytic Leukemia and Related B Cell Malignancies.

PubMed

Roberts, A W; Huang, Dcs

2017-01-01

The intracellular protein B-cell-lymphoma-2 (BCL2) has been considered an attractive target for cancer therapy since the discovery of its function as a major promoter of cell survival (an anti-apoptotic) in the late 1980s. However, the challenges of targeting a protein-protein interaction delayed the discovery of fit-for-purpose molecules until the mid-2000s. Since then, a series of high affinity small organic molecules that inhibits the interaction of BCL2 with the apoptotic machinery, the so-called BH3-mimetics, have been developed. Venetoclax (formerly ABT-199) is the first to achieve US Food and Drug Administration approval, with an indication for treatment of patients with previously treated chronic lymphocytic leukemia (CLL) bearing deletion of the long arm of chromosome 17. Here, we review key aspects of the science underpinning the clinical application of BCL2 inhibitors and explore both our current knowledge and unresolved questions about its clinical utility, both in CLL and in other B-cell malignancies that highly express BCL2. © 2016 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Differential ability of formononetin to stimulate proliferation of endothelial cells and breast cancer cells via a feedback loop involving MicroRNA-375, RASD1, and ERα.

PubMed

Chen, Jian; Zhang, Xing; Wang, Yong; Ye, Yu; Huang, Zhaoquan

2018-05-02

For postmenopausal cardiovascular disease, long-term estrogen therapy may increase the risk of breast cancer. To reduce this risk, estrogen may be replaced with the phytoestrogen formononetin, but how formononetin acts on vascular endothelial cells (ECs) and breast cancer cells is unclear. Here, we show that low concentrations of formononetin induced proliferation and inhibited apoptosis more strongly in cultured human umbilical vein endothelial cells (HUVECs) than in breast cancer cells expressing estrogen receptor α (ERα) (MCF-7, BT474) or not (MDA-MB-231), and that this differential stimulation was associated with miR-375 up-regulation in HUVECs. For the first time, we demonstrate the presence of a feedback loop involving miR-375, ras dexamethasone-induced 1 (RASD1), and ERα in normal HUVECs, and we show that formononetin stimulated this feedback loop in HUVECs but not in MCF-7 or BT474 cells. In all three cell lines, formononetin increased Akt phosphorylation and Bcl-2 expression. Inhibiting miR-375 blocked these changes and increased proliferation in HUVECs, but not in MCF-7 or BT474 cells. In ovariectomized rats, formononetin increased uterine weight and caused similar changes in levels of miR-375, RASD1, ERα, and Bcl-2 in aortic ECs as in cultured HUVECs. In mice bearing MCF-7 xenografts, tumor growth was stimulated by 17β-estradiol but not by formononetin. These results suggest selective action of formononetin in ECs (proliferation stimulation and apoptosis inhibition) relative to breast cancer cells, possibly via a feedback loop involving miR-375, RASD1, and ERα. This differential effect may explain why formononetin may not increase the risk of postmenopausal breast cancer. © 2018 Wiley Periodicals, Inc.

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

PubMed

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

2017-09-01

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-X L , 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-X L 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. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors.

PubMed

Yachida, Shinichi; Vakiani, Efsevia; White, Catherine M; Zhong, Yi; Saunders, Tyler; Morgan, Richard; de Wilde, Roeland F; Maitra, Anirban; Hicks, Jessica; Demarzo, Angelo M; Shi, Chanjuan; Sharma, Rajni; Laheru, Daniel; Edil, Barish H; Wolfgang, Christopher L; Schulick, Richard D; Hruban, Ralph H; Tang, Laura H; Klimstra, David S; Iacobuzio-Donahue, Christine A

2012-02-01

Poorly differentiated neuroendocrine carcinomas (NECs) of the pancreas are rare malignant neoplasms with a poor prognosis. The aim of this study was to determine the clinicopathologic and genetic features of poorly differentiated NECs and compare them with other types of pancreatic neoplasms. We investigated alterations of KRAS, CDKN2A/p16, TP53, SMAD4/DPC4, DAXX, ATRX, PTEN, Bcl2, and RB1 by immunohistochemistry and/or targeted exomic sequencing in surgically resected specimens of 9 small cell NECs, 10 large cell NECs, and 11 well-differentiated neuroendocrine tumors (PanNETs) of the pancreas. Abnormal immunolabeling patterns of p53 and Rb were frequent (p53, 18 of 19, 95%; Rb, 14 of 19, 74%) in both small cell and large cell NECs, whereas Smad4/Dpc4, DAXX, and ATRX labeling was intact in virtually all of these same carcinomas. Abnormal immunolabeling of p53 and Rb proteins correlated with intragenic mutations in the TP53 and RB1 genes. In contrast, DAXX and ATRX labeling was lost in 45% of PanNETs, whereas p53 and Rb immunolabeling was intact in these same cases. Overexpression of Bcl-2 protein was observed in all 9 small cell NECs (100%) and in 5 of 10 (50%) large cell NECs compared with only 2 of 11 (18%) PanNETs. Bcl-2 overexpression was significantly correlated with higher mitotic rate and Ki67 labeling index in neoplasms in which it was present. Small cell NECs are genetically similar to large cell NECs, and these genetic changes are distinct from those reported in PanNETs. The finding of Bcl-2 overexpression in poorly differentiated NECs, particularly small cell NEC, suggests that Bcl-2 antagonists/inhibitors may be a viable treatment option for these patients.

A leukemic double-hit follicular lymphoma associated with a complex variant translocation, t(8;14;18)(q24;q32;q21), involving BCL2, MYC, and IGH.

PubMed

Minakata, Daisuke; Sato, Kazuya; Ikeda, Takashi; Toda, Yumiko; Ito, Shoko; Mashima, Kiyomi; Umino, Kento; Nakano, Hirofumi; Yamasaki, Ryoko; Morita, Kaoru; Kawasaki, Yasufumi; Sugimoto, Miyuki; Yamamoto, Chihiro; Ashizawa, Masahiro; Hatano, Kaoru; Oh, Iekuni; Fujiwara, Shin-Ichiro; Ohmine, Ken; Kawata, Hirotoshi; Muroi, Kazuo; Miura, Ikuo; Kanda, Yoshinobu

2018-01-01

Double-hit lymphoma (DHL) is defined as lymphoma with concurrent BCL2 and MYC translocations. While the most common histological subtype of DHL is diffuse large B-cell lymphoma, the present patient had leukemic follicular lymphoma (FL). A 52-year-old man was admitted to our hospital due to general fatigue and cervical and inguinal lymph node swelling. The patient was leukemic and the pathological diagnosis of the inguinal lymph node was FL grade 1. Chromosomal analysis revealed a complex karyotype including a rare three-way translocation t(8;14;18)(q24;q32;q21) involving the BCL2, MYC, and IGH genes. Based on a combination of fluorescence in situ hybridization (FISH), using BCL2, MYC and IGH, and spectral karyotyping (SKY), the karyotype was interpreted as being the result of a multistep mechanism in which the precursor B-cell gained t(14;18) in the bone marrow and acquired a translocation between der(14)t(14;18) and chromosome 8 in the germinal center, resulting in t(8;14;18). The pathological diagnosis was consistently FL, not only at presentation but even after a second relapse. The patient responded well to standard chemotherapies but relapsed after a short remission. This patient is a unique case of leukemic DH-FL with t(8;14;18) that remained in FL even at a second relapse. Copyright © 2017 Elsevier Inc. All rights reserved.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

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.

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

ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity.

PubMed

Shao, Y; Aplin, A E

2012-01-19

B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only proteins represent a class of pro-apoptotic factors that neutralize pro-survival Bcl-2 proteins, and, in some cases, directly activate Bax. The mechanisms of control and the role of BH3-only proteins, such as Bcl-2 like protein 11 extra large and Bad are well studied. By contrast, relatively little is known about the regulation and role of Bcl-2 modifying factor (Bmf). The B-RAF oncogene is mutated in ∼8% of human tumors. We have previously shown that Bmf is upregulated at the transcript level and is required for apoptosis induced by targeting B-RAF signaling in tumor cells harboring mutant B-RAF. In this study, we show that Bmf is regulated at the post-translational level by mutant B-RAF-MEK-ERK2 signaling. Extracellular signal-regulated kinase (ERK2) directly phosphorylates Bmf on serine 74 and serine 77 residues with serine 77 being the predominant site. In addition, serine 77 phosphorylation reduces Bmf pro-apoptotic activity likely through a mechanism independent of altering Bmf localization to the mitochondria and/or interactions with dynein light chain 2 and the pro-survival proteins, B-cell lymphoma extra large, Bcl-2 and Mcl-1. These data identify a novel mode of regulation in Bmf that modulates its pro-apoptotic activity in mutant B-RAF tumor cells.

Rational combination strategies to enhance venetoclax activity and overcome resistance in hematologic malignancies.

PubMed

Grant, Steven

2018-06-01

Venetoclax (ABT-199) is a Bcl-2-specific BH3-mimetic that has shown significant promise in certain subtypes of CLL as well as in several other hematologic malignancies. As in the case of essentially all targeted agents, intrinsic or acquired resistance to this agent generally occurs, prompting the search for new strategies capable of circumventing this problem. A logical approach to this challenge involves rational combination strategies designed to disable preexisting or induced compensatory survival pathways. Many of these strategies involve downregulation of Mcl-1, a pro-survival Bcl-2 family member that is not targeted by venetoclax, and which often confers resistance to this agent. Given encouraging clinical results involving venetoclax in both lymphoid and myeloid malignancies, it is likely that such combination approaches will be incorporated into the therapeutic armamentarium for multiple hematologic malignancies in the near future.

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.

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

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

Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang

2012-03-01

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not onlymore » inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.« less

Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway

PubMed Central

Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng

2015-01-01

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia–reperfusion injury. PMID:26350953

Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

PubMed

Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

2009-07-01

Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

Therapeutic synergy between tigecycline and venetoclax in a preclinical model of MYC/BCL2 double-hit B cell lymphoma.

PubMed

Ravà, Micol; D'Andrea, Aleco; Nicoli, Paola; Gritti, Ilaria; Donati, Giulio; Doni, Mirko; Giorgio, Marco; Olivero, Daniela; Amati, Bruno

2018-01-31

High-grade B cell lymphomas with concurrent activation of the MYC and BCL2 oncogenes, also known as double-hit lymphomas (DHL), show dismal prognosis with current therapies. MYC activation sensitizes cells to inhibition of mitochondrial translation by the antibiotic tigecycline, and treatment with this compound provides a therapeutic window in a mouse model of MYC -driven lymphoma. We now addressed the utility of this antibiotic for treatment of DHL. BCL2 activation in mouse Eμ- myc lymphomas antagonized tigecycline-induced cell death, which was specifically restored by combined treatment with the BCL2 inhibitor venetoclax. In line with these findings, tigecycline and two related antibiotics, tetracycline and doxycycline, synergized with venetoclax in killing human MYC/BCL2 DHL cells. Treatment of mice engrafted with either DHL cell lines or a patient-derived xenograft revealed strong antitumoral effects of the tigecycline/venetoclax combination, including long-term tumor eradication with one of the cell lines. This drug combination also had the potential to cooperate with rituximab, a component of current front-line regimens. Venetoclax and tigecycline are currently in the clinic with distinct indications: Our preclinical results warrant the repurposing of these drugs for combinatorial treatment of DHL. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

BCL6 interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function

PubMed Central

Yang, Jessica A.; Tubo, Noah J.; Gearhart, Micah D.; Bardwell, Vivian J.; Jenkins, Marc K.

2015-01-01

CD4+ germinal center (GC) T follicular helper (GC-Tfh) cells help B cells become long-lived plasma cells and memory cells. The transcriptional repressor BCL6 plays a key role in GC-Tfh formation by inhibiting the expression of genes that promote differentiation into other lineages. We determined whether BCOR, a component of a Polycomb repressive complex that interacts with the BCL6 BTB domain, influences GC-Tfh differentiation. T cell-targeted BCOR deficiency led to a substantial loss of peptide:MHCII-specific GC-Tfh cells following Listeria monocytogenes infection and a 2-fold decrease following immunization with a peptide in CFA. The reduction in GC-Tfh cells was associated with diminished plasma cell and GC B cell formation. Thus, T cell-expressed BCOR is critical for optimal GC-Tfh differentiation and humoral immunity. PMID:25964495

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.

Rebamipide suppresses 5-fluorouracil-induced cell death via the activation of Akt/mTOR pathway and regulates the expression of Bcl-2 family proteins.

PubMed

Tsubaki, Masanobu; Takeda, Tomoya; Asano, Ryo-Ta; Matsuda, Tomoyuki; Fujimoto, Shin-Ichiro; Itoh, Tatsuki; Imano, Motohiro; Satou, Takao; Nishida, Shozo

2018-02-01

Oral mucositis is a common adverse effect of chemotherapy that limits the required dose of chemotherapeutic agents. Numerous attempts to mitigate chemotherapy-induced oral mucositis have failed to identify an appropriate treatment. Recently, it has been indicated that rebamipide prevents chemoradiotherapy-induced oral mucositis in patients. However, the details of the underlying mechanism involved in the cytoprotective effect of rebamipide remain obscure. In the present study, we investigated the mechanism behind rebamipide cytoprotective effect in the oral mucosa using primary normal human oral keratinocytes (NHOK cells). We found that rebamipide prevented 5-fluorouracil (5-FU)-induced cell death in NHOK cells. In addition, rebamipide increased the levels of phosphorylated Akt and mTOR, enhanced the Bcl-2 and Bcl-xL expressions, and suppressed the expression of Bax and Bim. This is in contrast to 5-FU-induced suppression of Akt and mTOR activation, Bcl-2 and Bcl-xL expressions, and the enhanced expression of Bax and Bim. These findings suggest that rebamipide can potentially be used for the protection of oral mucosa from chemotherapy-induced mucositis. This is the first study that elucidates the specific molecular pathway for the cytoprotective effect of rebamipide. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain

PubMed Central

Ashok, Iyaswamy; Sheeladevi, Rathinasamy

2014-01-01

Aspartame, an artificial sweetener, is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. Hence it is believed to be unsafe for human use. Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis. Hence, this present study is proposed to investigate whether or not chronic aspartame (FDA approved Daily Acceptable Intake (ADI),40 mg/kg bwt) administration could release methanol, and whether or not it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. To mimic the human methanol metabolism, Methotrexate (MTX)-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and catalase activity in (aspartame MTX)-treated animals and with a significant decrease in reduced glutathione, glutathione reductase and protein thiol, pointing out the generation of free radicals. The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain. PMID:25009784

Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain.

PubMed

Ashok, Iyaswamy; Sheeladevi, Rathinasamy

2014-01-01

Aspartame, an artificial sweetener, is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. Hence it is believed to be unsafe for human use. Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis. Hence, this present study is proposed to investigate whether or not chronic aspartame (FDA approved Daily Acceptable Intake (ADI),40 mg/kg bwt) administration could release methanol, and whether or not it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. To mimic the human methanol metabolism, Methotrexate (MTX)-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and catalase activity in (aspartame MTX)-treated animals and with a significant decrease in reduced glutathione, glutathione reductase and protein thiol, pointing out the generation of free radicals. The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain.

Apoptotic signaling pathways induced by acute administration of branched-chain amino acids in an animal model of maple syrup urine disease.

PubMed

Vilela, Thais C; Scaini, Giselli; Furlanetto, Camila B; Pasquali, Matheus A B; Santos, João Paulo A; Gelain, Daniel P; Moreira, José Cláudio F; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2017-02-01

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of the branched-chain α-keto acid dehydrogenase complex activity. This blockage leads to accumulation of the branched-chain amino acids leucine, isoleucine and valine, as well as their corresponding α-keto acids and α-hydroxy acids. The affected patients present severe neurological symptoms, such as coma and seizures, as well as edema and cerebral atrophy. Considering that the mechanisms of the neurological symptoms presented by MSUD patients are still poorly understood, in this study, protein levels of apoptotic factors are measured, such as Bcl-2, Bcl-xL, Bax, caspase-3 and -8 in hippocampus and cerebral cortex of rats submitted to acute administration of branched-chain amino acids during their development. The results in this study demonstrated that BCAA acute exposure during the early postnatal period did not significantly change Bcl-2, Bcl-xL, Bax and caspase-8 protein levels. However, the Bax/Bcl-2 ratio and procaspase-3 protein levels were decreased in hippocampus. On the other hand, acute administration of BCAA in 30-day-old rats increase in Bax/Bcl-2 ratio followed by an increased caspase-3 activity in cerebral cortex, whereas BCAA induces apoptosis in hippocampus through activation and cleavage of caspase-3 and -8 without changing the Bax/Bcl-2 ratio. In conclusion, the results suggest that apoptosis could be of pivotal importance in the developmental neurotoxic effects of BCAA. In addition, the current studies also suggest that multiple mechanisms may be involved in BCAA-induced apoptosis in the cerebral cortex and hippocampus.

Different effects of histone deacetylase inhibitors nicotinamide and trichostatin A (TSA) in C17.2 neural stem cells.

PubMed

Wang, Haifeng; Cheng, Hua; Wang, Kai; Wen, Tieqiao

2012-11-01

Histone deacetylase inhibitors are involved in proliferation, apoptosis, cell cycle, mRNA transcription, and protein expression in various cells. However, the molecular mechanism underlying such functions is still not fully clear. In this study, we used C17.2 neural stem cell (NSC) line as a model to evaluate the effects of nicotinamide and trichostatin A (TSA) on cell characteristics. Results show that nicotinamide and TSA greatly inhibit cell growth, lead to cell morphology changes, and effectively induce cell apoptosis in a dose-dependent manner. Western blot analyses confirmed that nicotinamide significantly decreases the expression of bcl-2 and p38. Further insight into the molecular mechanisms shows the suppression of phosphorylation in eukaryotic initiation factor 4E-binding protein 1 (4EBP1) by nicotinamide, whereas, an increased expression of bcl-2 and p38 and phosphorylation of 4EBP1 by TSA. However, both nicotinamide and TSA significantly increase the expression of cytochrome c (cyt c). These results strongly suggest that bcl-2, p38, cyt c, and p-4EBP1 could suppress proliferation and induce apoptosis of C17.2 NSCs mediated by histone deacetylase inhibitors, nicotinamide and TSA, involving different molecular mechanisms.

Comparative Evaluation of Silicone Hydrogel Contact Lenses and Autologous Serum for Management of Sjögren Syndrome-Associated Dry Eye.

PubMed

Li, Jinyang; Zhang, Xiahua; Zheng, Qinxiang; Zhu, Yirui; Wang, Hui; Ma, Huixiang; Jhanji, Vishal; Chen, Wei

2015-09-01

To comparatively evaluate the efficacy of a bandage contact lens (BCL) and autologous serum (AS) eye drops in the management of severe dry eye caused by Sjögren syndrome (SS). In this prospective randomized study, 40 patients with SS were enrolled. Patients were divided into 2 treatment groups: BCL and AS. A total of 37 patients were included, 18 patients (35 eyes) in the AS group and 19 patients (36 eyes) in the BCL group. At the end of 6 weeks, the best-corrected visual acuity improved significantly in the BCL group (0.5 ± 0.3 vs. 0.3 ± 0.2, P = 0.003) but not in the AS group (0.4 ± 0.3 vs. 0.3 ± 0.3, P = 0.11). The best-corrected visual acuity remained stable up to 6 weeks after discontinuation of the BCL (0.5 ± 0.3 vs. 0.4 ± 0.2, P = 0.03). Although the Ocular Surface Disease Index scores decreased significantly after treatment in both groups, patients in the BCL group had lower Ocular Surface Disease Index scores than those in the AS group (53.4 vs. 41.8 at week 3, 47.1 vs. 31.0 at week 6, 52.7 vs. 32.0 at week 12; P = 0.014, <0.001, <0.004, respectively). The "faces" scores showed improved quality of life in both groups. Tear break-up time improved significantly in both groups except at 6 weeks after discontinuation of the AS. Patients in the BCL group had lower corneal staining scores than those of the AS group after 6 weeks of treatment and 6 weeks after discontinuation of treatment (P < 0.01). There was no significant change in Schirmer I test scores between or within groups. Balafilcon A silicone hydrogel contact lenses as a BCL were effective in the management of SS-associated dry eye. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT02147509.

Computational Identification of Genomic Features That Influence 3D Chromatin Domain Formation.

PubMed

Mourad, Raphaël; Cuvier, Olivier

2016-05-01

Recent advances in long-range Hi-C contact mapping have revealed the importance of the 3D structure of chromosomes in gene expression. A current challenge is to identify the key molecular drivers of this 3D structure. Several genomic features, such as architectural proteins and functional elements, were shown to be enriched at topological domain borders using classical enrichment tests. Here we propose multiple logistic regression to identify those genomic features that positively or negatively influence domain border establishment or maintenance. The model is flexible, and can account for statistical interactions among multiple genomic features. Using both simulated and real data, we show that our model outperforms enrichment test and non-parametric models, such as random forests, for the identification of genomic features that influence domain borders. Using Drosophila Hi-C data at a very high resolution of 1 kb, our model suggests that, among architectural proteins, BEAF-32 and CP190 are the main positive drivers of 3D domain borders. In humans, our model identifies well-known architectural proteins CTCF and cohesin, as well as ZNF143 and Polycomb group proteins as positive drivers of domain borders. The model also reveals the existence of several negative drivers that counteract the presence of domain borders including P300, RXRA, BCL11A and ELK1.

Computational Identification of Genomic Features That Influence 3D Chromatin Domain Formation

PubMed Central

Mourad, Raphaël; Cuvier, Olivier

2016-01-01

Recent advances in long-range Hi-C contact mapping have revealed the importance of the 3D structure of chromosomes in gene expression. A current challenge is to identify the key molecular drivers of this 3D structure. Several genomic features, such as architectural proteins and functional elements, were shown to be enriched at topological domain borders using classical enrichment tests. Here we propose multiple logistic regression to identify those genomic features that positively or negatively influence domain border establishment or maintenance. The model is flexible, and can account for statistical interactions among multiple genomic features. Using both simulated and real data, we show that our model outperforms enrichment test and non-parametric models, such as random forests, for the identification of genomic features that influence domain borders. Using Drosophila Hi-C data at a very high resolution of 1 kb, our model suggests that, among architectural proteins, BEAF-32 and CP190 are the main positive drivers of 3D domain borders. In humans, our model identifies well-known architectural proteins CTCF and cohesin, as well as ZNF143 and Polycomb group proteins as positive drivers of domain borders. The model also reveals the existence of several negative drivers that counteract the presence of domain borders including P300, RXRA, BCL11A and ELK1. PMID:27203237

The Effect of Pentoxifylline on bcl-2 Gene Expression Changes in Hippocampus after Ischemia-Reperfusion in Wistar Rats by a Quatitative RT-PCR Method

PubMed Central

Sari, Soyar; Hashemi, Mehrdad; Mahdian, Reza; Parivar, Kazem; Rezayat, Mehdi

2013-01-01

Ischemia-reperfusion injury is the tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines. Twenty–four male Wistar rats (250-300 g body wt) were used in this study. The animals were divided into four groups of 6 rats each: I: Control group that was subjected to ischemia-reperfusion, II: Ischemia-reperfusion group that was subjected to all surgical procedures, III: Drug group that received pentoxifylline (200, 400 and 600 mg/kg) 60 min before and after ischemia and IV: Vehicle group that received saline. Seventy two h after ischemia-reperfusion, the hippocampus was taken for studying the changes in bcl-2 gene expression. We used quantitative real-time PCR for the detection of bcl-2 gene expression in ischemia and drug groups and then compared them to normal samples. The results showed the gene dosage ratio of 0.66 and 1.5 for ischemia group and the drug groups, respectively. The results also showed the bcl-2 gene expression declined in ischemia group as compared to the drug group. Furthermore, we observed a significant difference in the bcl-2 gene expression between ischemia and drug groups. These findings are consistent with anti-apoptotic properties of bcl-2 gene. Furthermore this method provides a powerful tool for the investigators to study brain ischemia and respond to the treatment drugs with anti-apoptotic agents. PMID:24250655

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

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

PubMed Central

Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

2015-01-01

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

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.

Low-energy BF2, BCl2, and BBr2 implants for ultrashallow P+-N junctions

NASA Astrophysics Data System (ADS)

Nandan, S. R.; Agarwal, Vikas; Banerjee, Sanjay K.

1997-08-01

We have examined low energy BCl2 and BBr2 implants as a means of fabricating ultra-shallow P+-N junctions. Five keV and 9 keV BCl2 implants and 18 keV BBr2 implants have been compared to 5 keV BF2 implants to study the benefits of using these species. BCl2 and BBr2, being heavier species, have a lower projected range and produce more damage. The greater damage restricts channeling, resulting in shallower as-implanted profiles. The increased damage amorphizes the substrate at low implant doses which results in reduced transient enhanced diffusion (TED) during the post-implant anneal. Post-anneal SIMS profiles indicate a junction depth reduction of over 10 nm (at 5 X 1017 cm-3 background doping) for 5 keV BCl2 implants as compared to 5 keV BF2 implants. Annealed junctions as shallow as 10 nm have been obtained from the 18 keV BBr2 implants. The increased damage degrades the electrical properties of these junctions by enhancing the leakage current densities. BCl2 implanted junctions have leakage current densities of approximately 1 (mu) A/cm2 as compared to 10 nA/cm2 for the BF2 implants. BBr2 implants have a lower leakage density of approximately 50 nA/cm2. Low energy BBr2 implants offer an exciting alternative for fabricating low leakage, ultra-shallow P+-N junctions.

Neuroprotection by baicalein in ischemic brain injury involves PTEN/AKT pathway.

PubMed

Liu, Chao; Wu, Jiliang; Xu, Kui; Cai, Fei; Gu, Jun; Ma, Liqun; Chen, Jianguo

2010-03-01

Recently more evidences support baicalein (Bai) is neuroprotective in models of ischemic stroke. This study was conducted to determine the molecular mechanisms involved in this effect. Either permanent or transient (2 h) middle cerebral artery occlusion (MCAO) was induced in rats in this study. Permanent MCAO led to larger infarct volumes in contrast to transient MCAO. Only in transient MCAO, Bai administration significantly reduced infarct size. Baicalein also markedly reduced apoptosis in the penumbra of transient MCAO rats. Additionally, oxygen and glucose deprivation (OGD) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular reactive oxygen species level and nitrotyrosine formation induced by OGD was counteracted by Bai, which is parallel with attenuated cell injury. The reduction of phosphorylation Akt and glycogen synthase kinase-3beta (GSK3beta) induced by OGD was restored by Bai, which was associated with preserved levels of phosphorylation of PTEN, the phophatase that negatively regulates Akt. As a consequence, Bcl-2/Bcl-xL-associated death protein phosphorylation was increased and the protein level of Bcl-2 in motochondria was maintained, which subsequently antagonize cytochrome c released in cytosol. LY294002 blocked the increase in phospho-AKT evoked by Bai and abolished the associated protective effect. Together, these findings provide evidence that Bai protects neurons against ischemia injury and this neuroprotective effect involves PI3K/Akt and PTEN pathway.

Strategic Therapeutic Targeting to Overcome Venetoclax Resistance in Aggressive B-cell Lymphomas.

PubMed

Pham, Lan V; Huang, Shengjian; Zhang, Hui; Zhang, Jun; Bell, Taylor; Zhou, Shouhao; Pogue, Elizabeth; Ding, Zhiyong; Lam, Laura; Westin, Jason; Davis, R Eric; Young, Ken H; Medeiros, L Jeffrey; Ford, Richard J; Nomie, Krystle; Zhang, Leo; Wang, Michael

2018-04-17

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 1-14. ©2018 AACR. ©2018 American Association for Cancer Research.

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 recurrent infection and vBcl-2-mediated survival of developing B cells. PMID:24516386

KRIBB11 accelerates Mcl-1 degradation through an HSF1-independent, Mule-dependent pathway in A549 non-small cell lung cancer cells.

PubMed

Kang, Min-Jung; Yun, Hye Hyeon; Lee, Jeong-Hwa

2017-10-21

The Bcl-2 family protein, Mcl-1 is known to have anti-apoptotic functions, and depletion of Mcl-1 by cellular stresses favors the apoptotic process. Moreover, Mcl-1 levels are frequently increased in various cancer cells, including non-small cell lung cancer (NSCLC), and is implicated in resistance to conventional chemotherapy and in cancer metastasis. In this study, we demonstrated that KRIBB11 accelerates the proteasomal degradation of Mcl-1 in the NSCLC cell line, A549. While KRIBB11 is an inhibitor of HSF1, we found that KRIBB11 induced Mcl-1 degradation in an HSF1-independent manner. Furthermore, this process was triggered via increase ubiquitination by the E3 ligase, Mule, rather than via de-ubiquitination by USP9X. Additionally, we found that Mcl-1 levels were only transiently reduced by KRIBB11: Mcl-1 levels were gradually restored as KRIBB11 activity diminished. However, we found that this effect was blocked in BIS (Bcl-2 interacting cell death suppressor, also called BAG3)-depleted cells, and that BIS prevents Mcl-1 from undergoing HSP70-driven proteasomal degradation, through an interaction with HSP70. Taken together, our results suggest that targeting Mcl-1 with KRIBB11 treatment, while simultaneously downregulating BIS, could be a therapeutic strategy in NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

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 induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl proteins was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and BCL2 i-Motif in Diffuse Large B-Cell Lymphoma Delays Tumor Growth.

PubMed

Kendrick, Samantha; Muranyi, Andrea; Gokhale, Vijay; Hurley, Laurence H; Rimsza, Lisa M

2017-08-10

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage.

PubMed

Xie, Chengzhi; Edwards, Holly; Caldwell, J Timothy; Wang, Guan; Taub, Jeffrey W; Ge, Yubin

2015-02-01

Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan-Bcl-2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double-strand breaks (DSBs) preceded a decrease of Mcl-1 levels, nuclear translocation of Bcl-2, Bcl-xL, and Mcl-1, and apoptosis. These results indicate that obatoclax enhances cytarabine-induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA-damaging agents in AML and possibly a broader range of malignancies. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

7 CFR 1717.604 - Long-range engineering plans and construction work plans.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 11 2010-01-01 2010-01-01 false Long-range engineering plans and construction work... AND GUARANTEED ELECTRIC LOANS Operational Controls § 1717.604 Long-range engineering plans and construction work plans. (a) All borrowers are required to maintain up-to-date long-range engineering plans and...

7 CFR 1717.604 - Long-range engineering plans and construction work plans.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 11 2013-01-01 2013-01-01 false Long-range engineering plans and construction work... AND GUARANTEED ELECTRIC LOANS Operational Controls § 1717.604 Long-range engineering plans and construction work plans. (a) All borrowers are required to maintain up-to-date long-range engineering plans and...

7 CFR 1717.604 - Long-range engineering plans and construction work plans.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 11 2014-01-01 2014-01-01 false Long-range engineering plans and construction work... AND GUARANTEED ELECTRIC LOANS Operational Controls § 1717.604 Long-range engineering plans and construction work plans. (a) All borrowers are required to maintain up-to-date long-range engineering plans and...

7 CFR 1717.604 - Long-range engineering plans and construction work plans.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 11 2012-01-01 2012-01-01 false Long-range engineering plans and construction work... AND GUARANTEED ELECTRIC LOANS Operational Controls § 1717.604 Long-range engineering plans and construction work plans. (a) All borrowers are required to maintain up-to-date long-range engineering plans and...

7 CFR 1717.604 - Long-range engineering plans and construction work plans.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 11 2011-01-01 2011-01-01 false Long-range engineering plans and construction work... AND GUARANTEED ELECTRIC LOANS Operational Controls § 1717.604 Long-range engineering plans and construction work plans. (a) All borrowers are required to maintain up-to-date long-range engineering plans and...

Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma.

PubMed

Chiron, David; Dousset, Christelle; Brosseau, Carole; Touzeau, Cyrille; Maïga, Sophie; Moreau, Philippe; Pellat-Deceunynck, Catherine; Le Gouill, Steven; Amiot, Martine

2015-04-20

The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL+MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-x(L) up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

Inducible nitric oxide synthase in T cells regulates T cell death and immune memory

PubMed Central

Vig, Monika; Srivastava, Smita; Kandpal, Usha; Sade, Hadassah; Lewis, Virginia; Sarin, Apurva; George, Anna; Bal, Vineeta; Durdik, Jeannine M.; Rath, Satyajit

2004-01-01

The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS+/+ APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS–/– T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination. PMID:15199408

Calotropin from Asclepias curasavica induces cell cycle arrest and apoptosis in cisplatin-resistant lung cancer cells.

PubMed

Mo, En-Pan; Zhang, Rong-Rong; Xu, Jun; Zhang, Huan; Wang, Xiao-Xiong; Tan, Qiu-Tong; Liu, Fang-Lan; Jiang, Ren-Wang; Cai, Shao-Hui

2016-09-16

Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity.

PubMed

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci.

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity

PubMed Central

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. PMID:27087825

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 targets to treat cancer. In the present review, we focused on the recent developments in therapeutic targeting of different steps in glycolysis, glutaminolysis and on the metabolic regulatory role of Bcl-2 family proteins.

Influence of long-term, high-dose dexamethasone administration on proliferation and apoptosis in porcine hepatocytes.

PubMed

Mikiewicz, Mateusz; Otrocka-Domagała, Iwona; Paździor-Czapula, Katarzyna; Rotkiewicz, Tadeusz

2017-06-01

The aim of this study was to examine the influence of long-term, high-dose dexamethasone administration on the liver, with particular emphasis on hepatocyte proliferation and apoptosis, using a swine model. The study included 48 large, female Polish breed pigs aged 3months (weighing ca. 30kg) divided into groups I (control; n=24) and II (dexamethasone; n=24) that receiving intra-muscular injections of monosodium phosphate dexamethasone for 29days. The pigs were euthanized on days subsequent to the experiment. Immediately after the euthanasia, the pig livers were sampled, fixed, and processed routinely for histopathology, histochemistry, and immunohistochemistry (for proliferating cell nuclear antigen, Bcl-2, and caspase-3). Apoptosis was visualized by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL). Dexamethasone administration gradually caused hepatocyte glycogen degeneration and finally lipid degeneration, accompanied by sinusoid and central vein dilatation and nuclear chromatin condensation. The proliferating cell nuclear antigen index, mean number of argyrophilic nucleolar organizer regions and proliferation index of argyrophilic nucleolar organizer regions were lower, while Bcl-2 expression was higher in group II compared with group I. The results from this study suggest that safe high-dose dexamethasone administration time is difficult to establish. Long-term, high-dose dexamethasone administration can cause pronounced morphological changes in hepatocytes by diminishing their transcriptional and proliferation activity but also protects them from apoptosis by potentially affecting Bcl-2 expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic inhibition of protein kinase Cε attenuates necrosis in experimental pancreatitis

PubMed Central

Liu, Yannan; Tan, Tanya; Jia, Wenzhuo; Lugea, Aurelia; Mareninova, Olga; Waldron, Richard T.; Pandol, Stephen J.

2014-01-01

Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis. PMID:25035113

Development and pilot line production of lithium doped silicon solar cells

NASA Technical Reports Server (NTRS)

Payne, P. A.

1972-01-01

The work performed over the period of September 1971 to August 1972 to develop production processes for fabrication of lithium doped P/N cells is described. The BCl3 diffusion without 02 was selected as the optimum diffusion process for fabrication of lithium doped cells. An 8-2-7 (warm up - deposition - drive-in time in minutes) diffusion schedule at 1055 C was used for the first two lots (300 cells each) delivered to JPL. Cell efficiencies ranged from 11.0 to 13.7% based on an AMO of 135.3 mW/sq cm. These high efficiencies were obtained using from 10 to 40 cells per boron diffusion; increasing the quantity beyond 40 resulted in lower outputs. At this point, the emphasis was placed on investigation of a BCl3 with 02 diffusion. Through evaluation of the effects of diffusion time and temperature, gas flow rates, and desposition plus drive-in vs. continuous deposition and no drive-in cycles, diffusion parameters were determined which produced short circuit currents of 136 + or - 4 mA for ten cells spaced along 12 in. of the diffusion boat. The quantity was increased to 60, 100, and 150 cell diffusions with no more variation in cell short circuit current than observed with 10 cells.

Heterolysis of H2 Across a Classical Lewis Pair, 2,6-Lutidine-BCl3: Synthesis, Characterization, and Mechanism

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

Ginovska-Pangovska, Bojana; Autrey, Thomas; Parab, Kshitij K.

We report on a combined computational and experimental study of the activation of hydrogen using for 2,6-lutidine (Lut)/BCl3 Lewis pairs. Herein we describe the synthetic approach used to obtain a new FLP, Lut-BCl3 that activates molecular H2 at ~10 bar, 100 °C in toluene or lutidine as the solvent. The resulting compound is an unexpected neutral hydride, LutBHCl2, rather than the ion pair, which we attribute to ligand redistribution. The mechanism for activation was modeled with density functional theory and accurate G3(MP2)B3 theory. The dative bond in Lut-BCl3 is calculated to have a bond enthalpy of 15 kcal/mol. The separatedmore » pair is calculated to react with H2 and form the [LutH+][HBCl3–] ion pair with a barrier of 13 kcal/mol. Metathesis with LutBCl3 produces LutBHCl2 and [LutH][BCl4]. The overall reaction is exothermic by 8.5 kcal/mol. An alternative pathway was explored involving lutidine–borenium cation pair activating H2. This work was supported by the U.S. Department of Energy's (DOE) Office of Basic Energy Sciences, Division of Chemical Sciences, Biosciences, and Geosciences, and was performed in part using the Molecular Science Computing Facility (MSCF) in the William R. Wiley Environmental Molecular Sciences Laboratory, a DOE national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for DOE.« less

Atherosclerosis-Associated Endothelial Cell Apoptosis by MiR-429-Mediated Down Regulation of Bcl-2.

PubMed

Zhang, Tao; Tian, Feng; Wang, Jing; Jing, Jing; Zhou, Shan-Shan; Chen, Yun-Dai

2015-01-01

Endothelial cell injury and subsequent apoptosis play a key role in the development and pathogenesis of atherosclerosis, which is hallmarked by dysregulated lipid homeostasis, aberrant immunity and inflammation, and plaque-instability-associated coronary occlusion. Nevertheless, our understanding of the mechanisms underlying endothelial cell apoptosis is still limited. MicroRNA-429 (miR-29) is a known cancer suppressor that promotes cancer cell apoptosis. However, it is unknown whether miR-429 may be involved in the development of atherosclerosis through similar mechanisms. We addressed these questions in the current study. We examined the levels of endothelial cell apoptosis in ApoE (-/-) mice suppled with high-fat diet (HFD), a mouse model for atherosclerosis (simplified as HFD mice). We analyzed the levels of anti-apoptotic protein Bcl-2 and the levels of miR-429 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-429 and 3'-UTR of Bcl-2 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-429 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs). HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/-) mice that had received normal diet (simplified as NOR mice) did not. HFD mice had significantly lower percentage of endothelial cells and significantly higher percentage of mesenchymal cells in the aorta than NOR mice. Significantly higher levels of endothelial cell apoptosis were detected in HFD mice, resulting from decreases in Bcl-2 protein, but not mRNA. The decreases in Bcl-2 in endothelial cells were due to increased levels of miR-429, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Atherosclerosis-associated endothelial cell apoptosis may result from down regulation of Bcl-2, through increased miR-429 that binds and suppresses translation of Bcl-2 mRNA. © 2015 The Author(s) Published by S. Karger AG, Basel.

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 downstream activation of caspases 9 and 3. However, the combination of DETANONOate and TRAIL resulted in activation of the mitochondrial pathway and activation of caspases 9 and 3, and induction of apoptosis. These findings demonstrate that DETANONOate-mediated sensitization of CaP to TRAIL-induced apoptosis is via inhibition of constitutive NF-kappa B activity and Bcl-(xL) expression.

Blood expression profiles of fragile X premutation carriers identify candidate genes involved in neurodegenerative and infertility phenotypes.

PubMed

Mateu-Huertas, Elisabet; Rodriguez-Revenga, Laia; Alvarez-Mora, Maria Isabel; Madrigal, Irene; Willemsen, Rob; Milà, Montserrat; Martí, Eulàlia; Estivill, Xavier

2014-05-01

Male premutation carriers presenting between 55 and 200 CGG repeats in the Fragile-X-associated (FMR1) gene are at risk of developing Fragile X Tremor/Ataxia Syndrome (FXTAS), and females undergo Premature Ovarian Failure (POF1). Here, we have evaluated gene expression profiles from blood in male FMR1 premutation carriers and detected a strong deregulation of genes enriched in FXTAS relevant biological pathways, including inflammation, neuronal homeostasis and viability. Gene expression profiling distinguished between control individuals, carriers with FXTAS and carriers without FXTAS, with levels of expanded FMR1 mRNA being increased in FXTAS patients. In vitro studies in a neuronal cell model indicate that expression levels of expanded FMR1 5'-UTR are relevant in modulating the transcriptome. Thus, perturbations of the transcriptome may be an interplay between the CGG expansion size and FMR1 expression levels. Several deregulated genes (DFFA, BCL2L11, BCL2L1, APP, SOD1, RNF10, HDAC5, KCNC3, ATXN7, ATXN3 and EAP1) were validated in brain samples of a FXTAS mouse model. Downregulation of EAP1, a gene involved in the female reproductive system physiology, was confirmed in female carriers. Decreased levels were detected in female carriers with POF1 compared to those without POF1, suggesting that EAP1 levels contribute to ovarian insufficiency. In summary, gene expression profiling in blood has uncovered mechanisms that may underlie different pathological aspects of the premutation. A better understanding of the transcriptome dynamics in relation with expanded FMR1 mRNA expression levels and CGG expansion size may provide mechanistic insights into the disease process and a more accurate FXTAS diagnosis to the myriad of phenotypes associated with the premutation. Copyright © 2014. Published by Elsevier Inc.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf

PubMed Central

Labi, Verena; Bertele, Daniela; Woess, Claudia; Tischner, Denise; Bock, Florian J; Schwemmers, Sven; Pahl, Heike L; Geley, Stephan; Kunze, Mirjam; Niemeyer, Charlotte M; Villunger, Andreas; Erlacher, Miriam

2013-01-01

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. ‘BH3-only’ proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34+ cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy. PMID:23180554

[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 bcl-2 gene translocation and ICN were found in 30 patients. Four ICNs of C-MYC gene were found in 28 patients. Elevated protein in cerebrospinal fluid (CSF) was found in 13 patients. LDH increased in 10 cases. Follow-up period was 2-90 months with the average survival time of (23.0±3.7) months and two-year survival rate of 39.0%. Univariate survival analysis showed that overexpression of bcl-2 protein (≥70%) and MYC protein (≥40%), bcl-2 gene abnormality (including copy number increase and translocation), C-MYC gene copy number increased were adverse factors for survival. C-MYC/ bcl-2 gene double hit was seen in 2 cases. Bivariate survival analysis found that of bcl-2/MYC protein double expression and bcl-2 and C-MYC genes double aberration were significantly associated with adverse outcomes. Cox multivariate risk regression analysis found that gender, cerebrospinal fluid protein increasing, and ICN of C-MYC gene were independent poor prognostic factors. DH-MTX based comprehensive chemotherapy was associated with better prognosis. Conclusions: Double hit at genomic level (copy number variations and gene rearrangements) and double protein expression of bcl-2 and C-MYC in PCNS-DLBCL are significantly associated with an adverse outcome. DH-MTX based comprehensive treatment may prolong the patient survival.

Docetaxel chronopharmacology in mice.

PubMed

Tampellini, M; Filipski, E; Liu, X H; Lemaigre, G; Li, X M; Vrignaud, P; François, E; Bissery, M C; Lévi, F

1998-09-01

Docetaxel tolerance and antitumor efficacy could be enhanced if drug administration was adapted to circadian rhythms. This hypothesis was investigated in seven experiments involving a total of 626 male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (12:12), after i.v. administration of docetaxel. In experiment (Exp) 1, the drug was given once a week (wk) for 6 wks (20 mg/kg/wk) or for 5 wks (30 mg/kg/wk) at one of six circadian times, during light when mice were resting [3, 7, or 11 hours after light onset (HALO)], or during darkness, when mice were active (15, 19, or 23 HALO). Endpoints were survival and body weight change. In Exp 2 and 3, docetaxel (30 mg/kg/wk) was administered twice, 1 wk apart, at one of four circadian stages (7, 11, 19, or 23 HALO). Endpoints were hematological and intestinal toxicities. In Exp 4, circadian changes in cell cycle phase distribution and BCL-2 immunofluorescence were investigated in bone marrow as possible mechanisms of docetaxel tolerability rhythm. In Exp 5 to 7, docetaxel was administered to mice bearing measurable P03 pancreatic adenocarcinoma (270-370 mg), with tumor weight and survival as endpoints. Mice from Exp 5 and 6 received a weekly schedule of docetaxel at one of six circadian stages (20 or 30 mg/kg/wk at 3, 7, 11, 15, 19, or 23 HALO). In Exp 7, docetaxel (30 mg/kg) was given every 2 days (day 1, 3, 5 schedule) at 7, 11, 19, or 23 HALO. Docetaxel dosing in the second half of darkness (19 or 23 HALO) resulted in significantly worse toxicity than its administration during the light span (3, 7, or 11 HALO). The survival rate ranged from 56.3% in the mice treated at 23 HALO to 93.8 or 87.5% in those injected at 3 or 11 HALO, respectively (Exp 1, P < 0.01). Granulocytopenia at nadir was -49 +/- 14% at 7 HALO compared with -84 +/- 3% at 19 HALO (Exp 2 and 3, P < 0.029), and severe jejunal mucosa necrosis occurred in 5 of 8 mice treated at 23 HALO as opposed to 2 of 18 receiving docetaxel at 7, 11, or 19 HALO (Exp 2 and 3, P < 0.02). The time of least docetaxel toxicity corresponded to the circadian nadir in S or G2-M phase and to the circadian maximum in BCL-2 immunofluorescence in bone marrow. Docetaxel increased the median survival of tumor-bearing mice in a dose-dependent manner (controls: 24 days; 20 mg/kg weekly, 33 days; 30 mg/kg weekly or day 1, 3, 5 schedule, 44 or 46 days, respectively; Exp 5-7). Survival curves of treated mice differed significantly according to dosing time for each dose and schedule (P from log rank <0.003 to P < 0.03). In Exp 5 and 6, the percentage of increase in life span was largest if docetaxel was administered weekly at 7 HALO (20 mg/kg, 220%; 30 mg/kg, 372%) and lowest after docetaxel dosing at 19 HALO (80% with 20 mg/kg) or at 15 HALO (78% with 30 mg/kg). In Exp 7, (day 1, 3, 5 schedule), docetaxel was most active at 11 HALO (percentage increase in life span, 390%) and least active at 23 HALO (210%). Docetaxel tolerability and antitumor efficacy were simultaneously enhanced by drug dosing in the light span, when mice were resting. Mechanisms underlying the tolerability rhythm likely involved the circadian organization of cell cycle regulation. Docetaxel therapeutic index may be improved with an administration at night in cancer patients, when fewest bone marrow cells are in S or G2-M phase.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

PubMed Central

Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris

2017-01-01

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255

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.

Curcumin Significantly Enhances Dual PI3K/Akt and mTOR Inhibitor NVP-BEZ235-Induced Apoptosis in Human Renal Carcinoma Caki Cells through Down-Regulation of p53-Dependent Bcl-2 Expression and Inhibition of Mcl-1 Protein Stability

PubMed Central

Cho, Il Je; Kim, Sang Chan; Kwon, Taeg Kyu

2014-01-01

The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level. PMID:24743574

The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

PubMed

Berrak, Özge; Akkoç, Yunus; Arısan, Elif Damla; Çoker-Gürkan, Ajda; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

2016-02-01

Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

MicroRNA-134-5p promotes high glucose-induced podocyte apoptosis by targeting bcl-2

PubMed Central

Qian, Xiaoxiao; Tan, Juan; Liu, Ling; Chen, Sheng; You, Na; Yong, Huijuan; Pan, Minglin; You, Qiang; Ding, Dafa; Lu, Yibing

2018-01-01

Podocyte apoptosis is a typical early feature of diabetic nephropathy (DN), with loss of nephrin integrity contributing to increased proteinuria in patients with DN. Emerging evidence shows that microRNAs (miRNAs) play vital roles in the pathogenesis of DN. Thus, we aimed to further elucidate the role of miRNAs in podocyte apoptosis in DN. We used db/db and db/m mice maintained under a continuous feeding regime for 12 weeks. Using microarray analysis, we found several miRNAs potentially related to podocyte apoptosis. In addition, we cultured a conditionally immortalized human podocyte cell line in 30 mM D-glucose and found that miR-134-5p was upregulated in both db/db mice and high-glucose (HG)-treated podocytes. Upregulation of miR-134-5p was accompanied by podocyte apoptosis and downregulation of nephrin. Inhibition of miR-134-5p produced the opposite effect. Dual-luciferase reporter assays showed that miR-134-5p directly targeted the 3’-untranslated region of the B-cell lymphoma-2 gene (BCL2), and further study confirmed an increase in bcl-2 protein level in HG-treated podocytes transfected with anti-miR-134-5p. Knockdown of BCL2 impeded the antiapoptotic effect of anti-miR-134-5p. Finally, we found that miR-134-5p might regulate apoptosis in db/db mice and podocytes by targeting BCL2. Taken together, our findings suggest that miR-134-5p promotes podocyte apoptosis under HG conditions by targeting BCL2. Our study provides a meaningful approach to interpret the mechanisms of action of miRNAs involved in DN. PMID:29636888

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53.

PubMed

Yang, Min-Chi; Lin, Ru-Wei; Huang, Shih-Bo; Huang, Shin-Yuan; Chen, Wen-Jie; Wang, Shiaw; Hong, Yi-Ren; Wang, Chihuei

2016-01-01

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.

Venetoclax: A new wave in hematooncology.

PubMed

Mihalyova, Jana; Jelinek, Tomas; Growkova, Katerina; Hrdinka, Matous; Simicek, Michal; Hajek, Roman

2018-05-01

Inhibitors of antiapoptotic proteins of the BCL2 family can successfully restart the deregulated process of apoptosis in malignant cells. Whereas nonselective agents have been limited by their affinity to different BCL2 members, thus inducing excessive toxicity, the highly selective BCL2 inhibitor venetoclax (ABT-199, Venclexta™) has an acceptable safety profile. To date, it has been approved in monotherapy for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL) with 17p deletion. Extension of indications can be expected in monotherapy and in combination regimens. Sensitivity to venetoclax is not common in lymphomas, but promising outcomes have been achieved in the mantle cell lymphoma group. Venetoclax is also active in multiple myeloma patients, especially in those with translocation t(11;14), even if high-risk features such as del17p are also present. Surprisingly, positive results are being obtained in elderly acute myeloid leukemia patients, in whom inhibition of BCL2 is able to substantially increase the efficacy of low-dose cytarabine or hypomethylating agents. Here, we provide a summary of available results from clinical trials and describe a specific mechanism of action that stands behind the efficacy of venetoclax in hematological malignancies. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Cell death pathways associated with PDT

NASA Astrophysics Data System (ADS)

Kessel, David; Reiners, John J., Jr.

2006-02-01

Photodynamic therapy leads to both direct and indirect tumor cell death. The latter also involves the consequences of vascular shut-down and immunologic effects. While these factors are a major factor in tumor eradication, there is usually an element of direct cell killing that can reduce the cell population by as much as 2-3 logs. Necrosis was initially believed to represent the predominant PDT death mechanism. An apoptotic response to PDT was first reported by Oleinick in 1991, using a sensitizer that targets the anti-apoptotic protein Bcl-2. Apoptosis leads to fragmentation of DNA and of cells into apoptotic bodies that are removed by phagocytosis. Inflammatory effects are minimized, and the auto- catalytic elements of the process can amplify the death signal. In this study, we examined consequences of Bcl-2 photodamage by a porphycene sensitizer that targets the ER and causes photodamage to the anti-apoptotic protein Bcl-2. Death patterns after Bcl-2 inactivation by a small-molecular antagonist were also assessed. In addition to apoptosis, we also characterized a hitherto undescribed PDT effect, the initiation of autophagy. Autophagy was initially identified as a cell survival pathway, allowing the recycling of components as nutrients become scarce. We propose that autophagy can also represent both a potential survival pathway after PDT damage to cellular organelles, as well as a cell-death pathway. Recent literature reports indicate that autophagy, as well as apoptosis, can be evoked after down-regulation of Bcl-2, a result consistent with results reported here.

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

PubMed

Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

2008-04-01

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

MicroRNA-10b downregulation mediates acute rejection of renal allografts by derepressing BCL2L11

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

Liu, Xiaoyou; Dong, Changgui; Jiang, Zhengyao

Kidney transplantation is the major therapeutic option for end-stage kidney diseases. However, acute rejection could cause allograft loss in some of these patients. Emerging evidence supports that microRNA (miRNA) dysregulation is implicated in acute allograft rejection. In this study, we used next-generation sequencing to profile miRNA expression in normal and acutely rejected kidney allografts. Among 75 identified dysregulated miRNAs, miR-10b was the most significantly downregulated miRNAs in rejected allografts. Transfecting miR-10b inhibitor into human renal glomerular endothelial cells recapitulated key features of acute allograft rejection, including endothelial cell apoptosis, release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor α, interferon-γ, andmore » chemokine (C–C motif) ligand 2) and chemotaxis of macrophages whereas transfection of miR-10b mimics had opposite effects. Downregulation of miR-10b directly derepressed the expression of BCL2L11 (an apoptosis inducer) as revealed by luciferase reporter assay. Taken together, miR-10b downregulation mediates many aspects of disease pathogenicity of acute kidney allograft rejection. Restoring miR-10b expression in glomerular endothelial cells could be a novel therapeutic approach to reduce acute renal allograft loss. - Highlights: • miR-10b was the most downregulated microRNAs in acutely rejected renal allografts. • miR-10b downregulation triggered glomerular endothelial cell apoptosis. • miR-10b downregulation induced release of pro-inflammatory cytokines. • miR-10b downregulation derepressed its pro-apoptotic target BCL2L11.« less

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.

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models.

PubMed

Punnoose, Elizabeth A; Leverson, Joel D; Peale, Franklin; Boghaert, Erwin R; Belmont, Lisa D; Tan, Nguyen; Young, Amy; Mitten, Michael; Ingalla, Ellen; Darbonne, Walter C; Oleksijew, Anatol; Tapang, Paul; Yue, Peng; Oeh, Jason; Lee, Leslie; Maiga, Sophie; Fairbrother, Wayne J; Amiot, Martine; Souers, Andrew J; Sampath, Deepak

2016-05-01

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL-selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2(High)/BCL-XL (Low) In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Ru(II)/diphenylphosphine/pyridine-6-thiolate complexes induce S-180 cell apoptosis through intrinsic mitochondrial pathway involving inhibition of Bcl-2 and p53/Bax activation.

PubMed

Pires, Wanessa Carvalho; Lima, Benedicto Augusto Vieira; de Castro Pereira, Flávia; Lima, Aliny Pereira; Mello-Andrade, Francyelli; Silva, Hugo Delleon; da Silva, Monize Martins; Colina-Vegas, Legna; Ellena, Javier; Batista, Alzir A; de Paul Silveira-Lacerda, Elisângela

2018-01-01

The aim of this work was the synthesis, characterization, and cytotoxicity evaluation of three new Ru(II) complexes with a general formula [Ru(Spy)(bipy)(P-P)]PF 6 [Spy = pyridine-6-thiolate; bipy = 2,2'-bipyridine; P-P = 1,2-bis(diphenylphosphine)ethane (1); 1,3-bis(diphenylphosphine) propane (2); and 1,1'-bis(diphenylphosphino)ferrocene] (4). Complex (3) with the 1,4-bis(diphenylphosphine)butane ligand, already known from the literature, was also synthesized, to be better studied here. The cytotoxicities of the complexes toward two kinds of cancerous cells (K562 and S-180 cells) were evaluated and compared to normal cells (L-929 and PBMC) by MTT assay. The complex [Ru(Spy)(bipy)(dppb)]PF 6 (3) was selected to study both the cellular and molecular mechanisms underlying its promising anticancer action in S-180 cells. The results obtained from this study indicated that complex (3) induces cell cycle arrest in the G0/G1 phase in S-180 cells associated with a decrease in the number of cells in S phase. After 24 and 48 h of exposure to complex (3), the cell viability decreased when compared to the negative control. Complex (3) does not appear to be involved in the DNA damage, but induced changes in the mitochondrial membrane potential in S-180 cells. Furthermore, there was also an increase in the gene expression of Bax, Caspase 9, and Tp53. According to our results, complex (3) induces cell apoptosis through p53/Bax-dependent intrinsic pathway and suppresses the expression of active antiapoptotic Bcl-2 protein.

CD10 down expression in follicular lymphoma correlates with gastrointestinal lesion involving the stomach and large intestine.

PubMed

Ohnishi, Nobuhiko; Takata, Katsuyoshi; Miyata-Takata, Tomoko; Sato, Yasuharu; Tari, Akira; Gion, Yuka; Noujima-Harada, Mai; Taniguchi, Kohei; Tabata, Tetsuya; Nagakita, Keina; Omote, Shizuma; Takahata, Hiroyuki; Iwamuro, Masaya; Okada, Hiroyuki; Maeda, Yoshinobu; Yanai, Hiroyuki; Yoshino, Tadashi

2016-11-01

Follicular lymphoma (FL) shows co-expression of B-cell lymphoma 2 (BCL2) and CD10, whereas downexpression of CD10 is occasionally experienced in gastrointestinal (GI) FL with unknown significance. Gastrointestinal FL is a rare variant of FL, and its similarity with mucosa-associated lymphoid tissue lymphoma was reported. We investigated the clinicopathological and genetic features of CD10 downexpressed (CD10 down ) GI-FL. The diagnosis of CD10 down FL was carried out with a combination of pathological and molecular analyses. The incidence of CD10 down GI-FL was shown in 35/172 (20.3%) cases, which was more frequent than nodal FL (3.5%, P < 0.001). The difference was additionally significant between GI-FL and nodal FL when the analysis was confined to primary GI-FL (55.2% vs 3.5%, P < 0.001). Compared to CD10 + GI-FL, CD10 down GI-FL significantly involved the stomach or large intestine (P = 0.015), and additionally showed the downexpression of BCL6 (P < 0.001). The follicular dendritic cell meshwork often showed a duodenal pattern in the CD10 down group (P = 0.12). Furthermore, a lymphoepithelial lesion was observed in 5/12 (40%) gastric FL cases, which indicated caution in the differentiation of mucosa-associated lymphoid tissue lymphoma. Molecular analyses were undertaken in seven cases of CD10 down GI-FL, and an identical clone was found between CD10 down follicles and CD10 + BCL2 + neoplastic follicles. In the diagnosis of cases with CD10 down BCL2 + follicles, careful examination with molecular studies should be carried out. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Sigma receptor 1 modulates ER stress and Bcl2 in murine retina.

PubMed

Ha, Yonju; Shanmugam, Arul K; Markand, Shanu; Zorrilla, Eric; Ganapathy, Vadivel; Smith, Sylvia B

2014-04-01

Sigma receptor 1 (σR1), a non-opiate transmembrane protein located on endoplasmic reticulum (ER) and mitochondrial membranes, is considered to be a molecular chaperone. Marked protection against cell death has been observed when ligands for σR1 have been used in in vitro and in vivo models of retinal cell death. Mice lacking σR1 (σR1(-/-)) manifest late-onset loss of retinal ganglion cells and retinal electrophysiological changes (after many months). The role of σR1 in the retina and the mechanisms by which its ligands afford neuroprotection are unclear. We therefore used σR1(-/-) mice to investigate the expression of ER stress genes (BiP/GRP78, Atf6, Atf4, Ire1α) and proteins involved in apoptosis (BCL2, BAX) and to examine the retinal transcriptome at young ages. Whereas no significant changes occurred in the expression of major ER stress genes (over a period of a year) in neural retina, marked changes were observed in these genes, especially Atf6, in isolated retinal Müller glial cells. BCL2 levels decreased in σR1(-/-) retina concomitantly with decreases in NFkB and pERK1/2. We postulate that σR1 regulates ER stress in retinal Müller cells and that the role of σR1 in retinal neuroprotection probably involves BCL2 and some of the proteins that modify its expression (such as ERK, NFκB). Data from the analysis of the retinal transcriptome of σR1 null mice provide new insights into the role of σR1 in retinal neuroprotection.

Sigma receptor 1 modulates ER stress and Bcl2 in murine retina

PubMed Central

Ha, Yonju; Shanmugam, Arul K.; Markand, Shanu; Zorrilla, Eric; Ganapathy, Vadivel; Smith, Sylvia B.

2014-01-01

Sigma receptor 1 (σR1), a non-opiate transmembrane protein located on endoplasmic reticulum (ER) and mitochondrial membranes, is considered a molecular chaperone. Marked protection against cell death has been observed when ligands for σR1 have been used in in vitro and in vivo models of retinal cell death. Mice lacking σR1 (σR1−/−) manifest late onset loss of retinal ganglion cells and retinal electrophysiological changes (after many months). The role of σR1 in retina and the mechanisms by which its ligands afford neuroprotection are unclear. To explore this we used σR1−/− mice and investigated expression of ER stress genes (BiP/GRP78, Atf6, Atf4, Ire1α) and proteins involved in apoptosis (BCL2, BAX) and examined the retinal transcriptome at young ages. While there were no significant changes in expression of major ER stress genes (over a period of a year) in neural retina, there were marked changes in these genes especially Atf6 in isolated retinal Müller glial cells. BCL2 levels decreased in σR1−/− retina concomitant with decreases in NFkB and pERK1/2. We postulate that σR1 regulates ER stress in retinal Müller cells and that the role of σR1 in retinal neuroprotection likely involves BCL2 and some of the proteins that modify its expression (such as ERK, NFκB). Data from the analysis of the retinal transcriptome of σR1 null mice provides new avenues to understand the role of σR1 in retinal neuroprotection. PMID:24469320

Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

PubMed Central

Drakopanagiotakis, Fotios; Xifteri, Areti; Tsiambas, Evaggelos; Karameris, Andreas; Tsakanika, Konstantina; Karagiannidis, Napoleon; Mermigkis, Demetrios; Polychronopoulos, Vlasis; Bouros, Demosthenes

2012-01-01

Introduction. Increased apoptosis of epithelial cells and decreased apoptosis of myofibroblasts are involved in the pathogenesis of IPF. The apoptotic profile of alveolar macrophages (AMs) in IPF is unclear. Aim. To investigate whether AMs of patients with IPF exhibit a different apoptotic profile compared to normal subjects. Methods. We analyzed, by immunohistochemistry, the expression of the apoptotic markers fas, fas ligand , bcl-2, and bax in AM obtained from bronchoalveolar lavage fluid (BALF) of 20 newly diagnosed, treatment-naive IPF patients and of 16 controls. Apoptosis of AM was evaluated by Apoptag immunohistochemistry. IPF patients received either interferon-g and corticosteroids or azathioprine and corticosteroids for six months. Results. BALF AMs undergoing apoptosis were significantly less in IPF patients. No difference was found in the expression of fas or fas ligand, bcl-2 and bax between IPF and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway. PMID:22792456

Short-Term Grafting of Human Neural Stem Cells: Electrophysiological Properties and Motor Behavioral Amelioration in Experimental Parkinsons Disease.

PubMed

Martnez-Serrano, Alberto; Pereira, Marta P; Avaliani, Natalia; Nelke, Anna; Kokaia, Merab; Ramos-Moreno, Tania

2016-12-13

Cell replacement therapy in Parkinsons disease (PD) still lacks a study addressing the acquisition of electrophysiological properties of human grafted neural stem cells and their relation with the emergence of behavioral recovery after transplantation in the short term. Here we study the electrophysiological and biochemical profiles of two ventral mesencephalic human neural stem cell (NSC) clonal lines (C30-Bcl-XL and C32-Bcl-XL) that express high levels of Bcl-XL to enhance their neurogenic capacity, after grafting in an in vitro parkinsonian model. Electrophysiological recordings show that the majority of the cells derived from the transplants are not mature at 6 weeks after grafting, but 6.7% of the studied cells showed mature electrophysiological profiles. Nevertheless, parallel in vivo behavioral studies showed a significant motor improvement at 7 weeks postgrafting in the animals receiving C30-Bcl-XL, the cell line producing the highest amount of TH+ cells. Present results show that, at this postgrafting time point, behavioral amelioration highly correlates with the spatial dispersion of the TH+ grafted cells in the caudate putamen. The spatial dispersion, along with a high number of dopaminergic-derived cells, is crucial for behavioral improvements. Our findings have implications for long-term standardization of stem cell-based approaches in Parkinsons disease.

Micro-Economics of Apoptosis in Cancer: ncRNAs Modulation of BCL-2 Family Members

PubMed Central

Villanova, Lidia; Careccia, Silvia; De Maria, Ruggero

2018-01-01

In the last few years, non-coding RNAs (ncRNAs) have been a hot topic in cancer research. Many ncRNAs were found to regulate the apoptotic process and to play a role in tumor cell resistance to treatment. The apoptotic program is on the frontline as self-defense from cancer onset, and evasion of apoptosis has been classified as one of the hallmarks of cancer responsible for therapy failure. The B-cell lymphoma 2 (BCL-2) family members are key players in the regulation of apoptosis and mediate the activation of the mitochondrial death machinery in response to radiation, chemotherapeutic agents and many targeted therapeutics. The balance between the pro-survival and the pro-apoptotic BCL-2 proteins is strictly controlled by ncRNAs. Here, we highlight the most common mechanisms exerted by microRNAs, long non-coding RNAs and circular RNAs on the main mediators of the intrinsic apoptotic cascade with particular focus on their significance in cancer biology. PMID:29570632

Micro-Economics of Apoptosis in Cancer: ncRNAs Modulation of BCL-2 Family Members.

PubMed

Villanova, Lidia; Careccia, Silvia; De Maria, Ruggero; Fiori, Micol E

2018-03-23

In the last few years, non-coding RNAs (ncRNAs) have been a hot topic in cancer research. Many ncRNAs were found to regulate the apoptotic process and to play a role in tumor cell resistance to treatment. The apoptotic program is on the frontline as self-defense from cancer onset, and evasion of apoptosis has been classified as one of the hallmarks of cancer responsible for therapy failure. The B-cell lymphoma 2 (BCL-2) family members are key players in the regulation of apoptosis and mediate the activation of the mitochondrial death machinery in response to radiation, chemotherapeutic agents and many targeted therapeutics. The balance between the pro-survival and the pro-apoptotic BCL-2 proteins is strictly controlled by ncRNAs. Here, we highlight the most common mechanisms exerted by microRNAs, long non-coding RNAs and circular RNAs on the main mediators of the intrinsic apoptotic cascade with particular focus on their significance in cancer biology.

Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism.

PubMed

Luanpitpong, Sudjit; Nimmannit, Ubonthip; Chanvorachote, Pithi; Leonard, Stephen S; Pongrakhananon, Varisa; Wang, Liying; Rojanasakul, Yon

2011-08-01

Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.

Glucocorticoid sensitisation in Mixed Lineage Leukaemia-rearranged acute lymphoblastic leukaemia by the pan-BCL-2 family inhibitors gossypol and AT-101.

PubMed

Spijkers-Hagelstein, Jill A P; Schneider, Pauline; Pinhanços, Sandra Mimoso; Garrido Castro, Patricia; Pieters, Rob; Stam, Ronald W

2014-06-01

Resistance to glucocorticoids (GCs) remains a major problem in the treatment of infants with acute lymphoblastic leukaemia (ALL) carrying Mixed Lineage Leukaemia (MLL) translocations. Despite intensive research, the mechanism(s) underlying GC resistance remain poorly understood. Recent studies suggested an important role for the pro-survival BCL-2 family member MCL1 in GC resistance in MLL-rearranged ALL. We exposed GC-resistant MLL-rearranged SEMK2 cells to potent MCL1-inhibiting agents, including gossypol, AT-101, rapamycin, SU9516 and obatoclax (GX15-070) and determined GC sensitisation using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays. Using Western blotting we analysed the protein expression of most BCL-2 family members in MLL-rearranged SEMK2 cells after treatment with potent MCL-1 inhibiting agents. Only gossypol and its synthetic analogue AT-101 induced GC sensitivity in MLL-rearranged ALL cells. Remarkably, the GC-sensitising effects of gossypol and AT-101 appeared not to be mediated by down-regulation MCL1 or other anti-apoptotic BCL-2 family members, but rather involved up-regulation of multiple pro-apoptotic BCL-2 family members, in particular that of BIM and BID. In conclusion, gossypol and AT-101 induce GC sensitivity in MLL-rearranged ALL cells, most likely mediated by the activation of BID and BIM without the necessity to down-regulate anti-apoptotic BCL-2 family members like MCL1. Hence, co-administration of either gossypol or AT-101 during GC treatment of GC-resistant MLL-rearranged ALL patients may overcome GC resistance and improve prognosis in this high-risk childhood leukaemia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Propofol improved neurobehavioral outcome of cerebral ischemia-reperfusion rats by regulating Bcl-2 and Bax expression.

PubMed

Xi, Hong-Jie; Zhang, Tian-Hua; Tao, Tao; Song, Chun-Yu; Lu, Shu-Jun; Cui, Xiao-Guang; Yue, Zi-Yong

2011-09-02

Propofol is an intravenous anesthetic with neuroprotective effects against cerebral ischemia-reperfusion (I/R) injury. Few studies regarding the neuroprotective and neurobehavioral effects of propofol have been conducted, and the underlying mechanisms are still unclear. Because I/R may result in neuronal apoptosis, the apoptosis regulatory genes B-cell leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax) may be involved in the neuroprotective process. In this study, 120 Wistar rats were randomly divided into three groups (sham, I/R-induced, and propofol-treated). Cerebral ischemia was induced by clamping the bilateral common carotid arteries for 10min. Propofol (1.0mg/kg/min) was administered intravenously for 1h before the induction of ischemia. Neuronal damage was evaluated by neurobehavioral scores and histological examination of the brain sections at the level of the dorsal hippocampus at 6h, 24h, 48h, 72h, 4days, 5days, 6days, and 7days after I/R. The apoptotic rate of hippocampal neurons was detected by flow cytometry. The expression of Bcl-2 and Bax was evaluated using immunohistochemical and Western blot methods. The results of this study showed that neurobehavioral scores were higher in propofol-treated rats compared with I/R-induced rats with no propofol treatment. Moreover, the hippocampal expression of Bcl-2 was significantly higher, while the expression of Bax was significantly lower in propofol-treated rats compared with I/R-induced rats at 24h after ischemia. Hence, this study suggests that the neuroprotective effects of propofol against neuronal apoptosis may be a consequence of the regulation of Bcl-2 and Bax. Copyright © 2011 Elsevier B.V. All rights reserved.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

PubMed

Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

2017-01-01

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

Molecular Characterization of Acquired Tolerance of Tumor Cells to Picropodophyllin (PPP)

PubMed Central

Hashemi, Jamileh; Worrall, Claire; Vasilcanu, Daiana; Fryknäs, Mårten; Sulaiman, Luqman; Karimi, Mohsen; Weng, Wen-Hui; Lui, Weng-Onn; Rudduck, Christina; Axelson, Magnus; Jernberg-Wiklund, Helena; Girnita, Leonard; Larsson, Olle; Larsson, Catharina

2011-01-01

Background Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Methodology/Principal Findings Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/ amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Conclusions Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions. PMID:21423728

Molecular characterization of acquired tolerance of tumor cells to picropodophyllin (PPP).

PubMed

Hashemi, Jamileh; Worrall, Claire; Vasilcanu, Daiana; Fryknäs, Mårten; Sulaiman, Luqman; Karimi, Mohsen; Weng, Wen-Hui; Lui, Weng-Onn; Rudduck, Christina; Axelson, Magnus; Jernberg-Wiklund, Helena; Girnita, Leonard; Larsson, Olle; Larsson, Catharina

2011-03-14

Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.

The Effects of Cadmium at Low Environmental Concentrations on THP-1 Macrophage Apoptosis

PubMed Central

Olszowski, Tomasz; Baranowska-Bosiacka, Irena; Gutowska, Izabela; Piotrowska, Katarzyna; Mierzejewska, Katarzyna; Korbecki, Jan; Kurzawski, Mateusz; Tarnowski, Maciej; Chlubek, Dariusz

2015-01-01

Cadmium at environmental concentrations is a risk factor for many diseases, including cardiovascular and neurodegenerative diseases, in which macrophages play an important role. The aim of this study was to evaluate the effects of cadmium at low environmental (nanomolar) concentrations on apoptotic processes in THP-1(acute monocytic leukemia cells line)-derived macrophages, with special focus on mitochondrial events involved. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM and 2 µM CdCl2. Cell viability was measured using flow cytometry. Flow cytometric measurement (annexin V/FITC (annexin V/fluorescein isothiocyanate) and PI (propidium iodide) double staining) was used to quantify the extent of apoptosis. Fluorescence and confocal microscopy were used for imaging of apoptosis process. Changes in mitochondrial membrane potential were monitored using cytofluorimetry after cell staining with JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyane iodide) probe. Mitochondrial ROS (reactive oxygen species) levels were measured cytofluorimetrically after incubation of cells with mitochondrial superoxide indicator (MitoSOX) red fluorescent marker. The mRNA expression of Bcl-2 and Bax was analysed with qRT-PCR. Our study demonstrates that cadmium, even at low environmental concentrations, exerts mitochondrial toxicity in THP-1 macrophages. Forty-eight-hour exposure to very low concentrations reduces cell viability and results in cell death by apoptosis and necrosis. The decrease in mitochondrial membrane potential, increased ROS production, increased Bax and decreased Bcl-2 mRNA expression are mitochondrial events involved in cadmium-induced apoptosis. PMID:26370970

Rasagiline delays retinal degeneration in a mouse model of retinitis pigmentosa via modulation of Bax/Bcl-2 expression.

PubMed

Garcia-Delgado, Ana B; Valdés-Sánchez, Lourdes; Calado, Sofia M; Diaz-Corrales, Francisco J; Bhattacharya, Shom S

2018-05-01

Retinitis pigmentosa (RP) is an inherited disease characterized by a progressive degeneration of rod photoreceptors. An imbalance between pro- and antiapoptotic factors, such as Bax/Bcl-2, has been involved in retinal degeneration. To date, no cure or effective treatments are available for RP. Rasagiline is an antiparkinsonian drug that has shown neuroprotective effects in part attributed to a modulation of Bax/Bcl-2 expression. In this study, we have evaluated the use of rasagiline as a potential treatment for RP. Newborn rd10 mice, a RP model, were treated with oral rasagiline during 30 days followed by a functional and morphological characterization of their mouse retinas. Treated animals showed a significant improvement in visual acuity and in the electrical responses of photoreceptors to light stimuli. Rasagiline delayed photoreceptor degeneration, which was confirmed not only by a high photoreceptor nuclei counting, but also by a sustained expression of photoreceptor-specific markers. In addition, the expression of proapoptotic Bax decreased, whereas the antiapoptotic factor Bcl-2 increased after rasagiline treatment. This study provides new evidences regarding the neuroprotective effect of rasagiline in the retina, and it brings new insight into the development of future clinical trials using this well-established antiparkinsonian drug to treat RP. © 2017 John Wiley & Sons Ltd.

A novel synthetic compound exerts effective anti-tumour activity in vivo via the inhibition of tubulin polymerisation in A549 cells.

PubMed

Yan, Jun; Pang, Yanqing; Sheng, Jianfeng; Wang, Yali; Chen, Jie; Hu, Jinhui; Huang, Ling; Li, Xingshu

2015-09-01

Microtubules are critical elements that are involved in a wide range of cellular processes, and thus, they have become an attractive target for many anticancer drugs. A novel synthesised compound, 12P, was identified as new microtubule inhibitor. This compound inhibits tubulin polymerisation through binding to the colchicine-binding site of tubulin. 12P exhibits excellent anti-proliferative activities against a panel of human cancer cell lines, with IC₅₀ values range from 9 to 55nM. Interestingly, compound 12P also displayed equally potent cytotoxicity against several drug-resistant cell lines, and it showed high selectivity for active human umbilical vein endothelial cells (HUVECs). Further flow cytometric analysis showed that 12P induces G₂/M phase arrest and apoptosis in A549 cells. Cellular studies have revealed that the induction of apoptosis by 12P was associated with a collapse of mitochondrial membrane potential (MMP), accumulation of reactive oxygen species (ROS), alterations in the expression of some cell cycle-related proteins (e.g. Cyclin B1, Cdc25c, Cdc2) and some apoptosis-related proteins (e.g. Bax, Bad, Bcl-2, Bcl-xl). Importantly, 12P significantly reduced the growth of xenograft tumours of A549 cells in vivo (tumour inhibitory rate of 12P: 84.2%), without any loss of body weight. Taken together, these in vitro and in vivo results suggested that 12P may become a promising lead compound for the development of new anticancer drugs. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

PubMed

Renault, Thibaud T; Floros, Konstantinos V; Elkholi, Rana; Corrigan, Kelly-Ann; Kushnareva, Yulia; Wieder, Shira Y; Lindtner, Claudia; Serasinghe, Madhavika N; Asciolla, James J; Buettner, Christoph; Newmeyer, Donald D; Chipuk, Jerry E

2015-01-08

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic and physical map of the von Recklinghausen neurofibromatosis (NF1) region on chromosome 17

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

Yagle, M.K.; Parruti, G.; Xu, W.

The von Recklinghausen neurofibromatosis 1 (NF1) locus has been previously assigned to the proximal long arm of chromosome 17, and two NF1 patients have been identified who have constitutional balanced translocations involving 17q11.2. The authors have constructed a cosmid library from a chromosome-mediated gene transfectant, KLT8, that contains approximately 10% of chromosome 17, including 17q11.2. Cosmids isolated from this library have been mapped across a panel of somatic cell hybrids, including the hybrids from the two patients, and have been localized to seven small regions of proximal 17q. They have 5 cosmids that map directly above the two NF1 translocations,more » and 11 cosmids that map directly below. Of these, 2 cosmids in each region are linked to the disease locus and 3 of these cosmids show no recombination. One distal cosmid, 2B/B35, detects the two NF1 translocations by pulsed-field gel analysis and has been used to produce a long-range restriction map that covers the translocations.« less

Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions.

PubMed

Fan, Xiaorui; Xi, Huaming; Zhang, Zhen; Liang, Yajun; Li, Qinghong; He, Junping

2017-04-01

The aim of this study was to examine whether an elevated ambient temperature (37-40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37-40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20-27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (P<0.01), and the cell types most affected by heat treatment were spermatocytes and spermatids. In both the control and experimental groups, Bcl-2 was expressed in the cytoplasm and nucleus of spermatogonia, spermatocytes and differentiating spermatids and Bcl-2 preferentially localized close to the seminiferous tubule's luminal surface in late spermatocytes and spermatids. Compared with the control group, the expression levels of Bcl-2 protein and mRNA significantly increased in heat treatment group, while the expression levels of Bax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars. Copyright © 2016. Published by Elsevier GmbH.

Time-Resolved Quantum Cascade Laser Absorption Spectroscopy of Pulsed Plasma Assisted Chemical Vapor Deposition Processes Containing BCl3

NASA Astrophysics Data System (ADS)

Lang, Norbert; Hempel, Frank; Strämke, Siegfried; Röpcke, Jürgen

2011-08-01

In situ measurements are reported giving insight into the plasma chemical conversion of the precursor BCl3 in industrial applications of boriding plasmas. For the online monitoring of its ground state concentration, quantum cascade laser absorption spectroscopy (QCLAS) in the mid-infrared spectral range was applied in a plasma assisted chemical vapor deposition (PACVD) reactor. A compact quantum cascade laser measurement and control system (Q-MACS) was developed to allow a flexible and completely dust-sealed optical coupling to the reactor chamber of an industrial plasma surface modification system. The process under the study was a pulsed DC plasma with periodically injected BCl3 at 200 Pa. A synchronization of the Q-MACS with the process control unit enabled an insight into individual process cycles with a sensitivity of 10-6 cm-1·Hz-1/2. Different fragmentation rates of the precursor were found during an individual process cycle. The detected BCl3 concentrations were in the order of 1014 molecules·cm-3. The reported results of in situ monitoring with QCLAS demonstrate the potential for effective optimization procedures in industrial PACVD processes.

Long non-coding RNA ANRIL is up-regulated in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic pathway

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

Zhu, Hongxue; Department of Urology, Hospital of Xinjiang Production and Construction Corps, Urumqi 830002; Li, Xuechao

Antisense non-coding RNA in the INK4 locus (ANRIL) is a member of long non-coding RNAs and has been reported to be dysregulated in several human cancers. However, the role of ANRIL in bladder cancer remains unclear. This present study aimed to investigate whether and how ANRIL involved in bladder cancer. Our results showed up-regulation of ANRIL in bladder cancer tissues versus the corresponding adjacent non-tumor tissues. To explore the specific mechanisms, ANRIL was silenced by small interfering RNA or short hairpin RNA transfection in human bladder cancer T24 and EJ cells. Knockdown of ANRIL repressed cell proliferation and increased cellmore » apoptosis, along with decreased expression of Bcl-2 and increased expressions of Bax, cytoplasmic cytochrome c and Smac and cleaved caspase-9, caspase-3 and PARP. However, no change of cleaved caspase-8 level was observed. Furthermore, in vivo experiment confirmed that knockdown of ANRIL inhibited tumorigenic ability of EJ cells in nude mice. Meanwhile, in accordance with in vitro study, knockdown of ANRIL inhibited expression of Bcl-2 and up-regulated expressions of Bax and cleaved caspase-9, but did not affect cleaved caspase-8 level. In conclusion, we first report that ANRIL possibly serves as an oncogene in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic apoptosis pathway. - Highlights: • We first report the role of ANRIL in bladder cancer. • ANRIL is obviously up-regulated in bladder cancer tissues. • ANRIL regulates bladder cancer cell proliferation and cell apoptosis through the intrinsic pathway.« less

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

PubMed

Arsov, I; Li, X; Matthews, G; Coradin, J; Hartmann, B; Simon, A K; Sealfon, S C; Yue, Z

2008-09-01

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

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

Quinolone analogue inhibits tubulin polymerization and induces apoptosis via Cdk1-involved signaling pathways.

PubMed

Chen, Ying-Cheng; Lu, Pin-Hsuan; Pan, Shiow-Lin; Teng, Che-Ming; Kuo, Sheng-Chu; Lin, Tsung-Ping; Ho, Yunn-Fang; Huang, Yu-Chun; Guh, Jih-Hwa

2007-06-30

Cancer chemotherapeutic agents that interfere with tubulin/microtubule function are in extensive use. Quinolone is a common structure in alkaloids and its related components exhibit several pharmacological activities. In this study, we have identified the anticancer mechanisms of 2-phenyl-4-quinolone. 2-Phenyl-4-quinolone displayed anti-proliferative effect in several cancer types, including hormone-resistant prostate cancer PC-3, hepatocellular carcinoma Hep3B and HepG2, non-small cell lung cancer A549 and P-glycoprotein-rich breast cancer NCI/ADR-RES cells. The IC(50) values were 0.85, 1.81, 3.32, 0.90 and 1.53 microM, respectively. 2-Phenyl-4-quinolone caused G2/M arrest of the cell-cycle and a subsequent apoptosis. The turbidity assay showed an inhibitory effect on tubulin polymerization. After immunochemical examination, the data demonstrated that the microtubules were arranged irregularly into dipolarity showing prometaphase-like states. Furthermore, 2-Phenyl-4-quinolone induced the Mcl-1 cleavage, the phosphorylation of Bcl-2 and Bcl-xL (12-h treatment), and the caspase activation including caspase-8, -2 and -3 (24-h treatment). The exposure of cells to 2-phenyl-4-quinolone caused Cdk1 activation by several observations, namely (i) elevation of cyclin B1 expression, (ii) dephosphorylation on inhibitory Tyr-15 of Cdk1, and (iii) dephosphorylation on Ser-216 of Cdc25c. Moreover, a long-term treatment (36h) caused the release reaction and subsequent nuclear translocation of AIF. In summary, it is suggested that 2-phenyl-4-quinolone displays anticancer effect through the dysregulation of mitotic spindles and induction of mitotic arrest. Furthermore, participation of cell-cycle regulators, Bcl-2 family of proteins, activation of caspases and release of AIF may mutually cross-regulate the apoptotic signaling cascades induced by 2-phenyl-4-quinolone.

A Caspase-Resistant Form of Bcl-XL, but Not Wild Type Bcl-XL, Promotes Clonogenic Survival After Ionizing Radiation

PubMed Central

Rehemtulla, Alnawaz; Hamilton, A Christin; Taneja, Neelam; Fridman, Jordan; Juan, Todd SC; Maybaum, Jonathan; Chinnaiyan, Arul

1999-01-01

Abstract Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL Bcl-XLΔloop. Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy. PMID:10935471

Activated carbon N-acetylcysteine microcapsule protects against nonalcoholic fatty liver disease in young rats via activating telomerase and inhibiting apoptosis

PubMed Central

Zhou, Hongping; Xi, Jianjun; Sun, Jingjing; Ke, Yunling; Zhang, Jiankang; Shao, Yidan; Jiang, Xiaojie; Pan, Xuwang; Liu, Shourong; Zhuang, Rangxiao

2018-01-01

Non-alcoholic fatty liver disease (NAFLD) is becoming one of the world's most common chronic liver diseases in childhood, yet no therapy is available that has been approved by the food and drug administration (FDA). Previous studies have reported that telomere and telomerase are involved the development and progression of NAFLD. This study was designed to investigate the potential beneficial effects of activated carbon N-acetylcysteine (ACNAC) microcapsules on the development of NAFLD in young rats as well as the underlying mechanism(s) involved. Three-week old male Sprague Dawley rats were given high-fat diet (HFD) with/without ACNAC treatment for 7 consecutive weeks. Liver pathologies were determined by hematoxylin and eosin (H&E) and Oil Red O staining, as well as by changes in biochemical parameters of plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, respectively. Glucose homeostasis was evaluated by the glucose tolerance test and the liver telomere length and activity were measured by real time PCR and telomeric repeat amplification protocol (TRAP). Western blot analysis was performed to determine the expression level of Bcl-2, Bax and Caspase-3. Our results demonstrated that ACNAC supplementation improved liver pathologies of rats that received long-term HFD feeding. ACNAC supplementation prevented HFD-induced telomere shortening and improved telomerase activity. Moreover, in comparison to HFD-fed rats, ACNAC supplementation markedly increased the expression of Bcl-2, but significantly decreased the expression of Bax and Caspase-3 in juvenile rats. Together, these results indicate that ACNAC may be a promising choice for preventing and treating NAFLD among children. PMID:29324774

Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

PubMed

Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

2015-04-01

The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p < 0.001). IL-8 productions from GES-1 cells infected with the wild type strain were significantly higher than from those with the mutant (p < 0.001). The amounts of vital MKN-45 cells were decreased and the numbers of apoptotic cells were increased with the wild type strain, compared to those with the mutant after 12 h (p < 0.05). The increase of cleaved Caspase-3 and Bax was significantly higher and the decrease of Bcl-2 was more obvious in MKN-45 cells exposed to the wild type strain than that exposed to the mutant after 6 h. We demonstrate that intact long-type DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Large-scale genome-wide association studies in East Asians identify new genetic loci influencing metabolic traits.

PubMed

Kim, Young Jin; Go, Min Jin; Hu, Cheng; Hong, Chang Bum; Kim, Yun Kyoung; Lee, Ji Young; Hwang, Joo-Yeon; Oh, Ji Hee; Kim, Dong-Joon; Kim, Nam Hee; Kim, Soeui; Hong, Eun Jung; Kim, Ji-Hyun; Min, Haesook; Kim, Yeonjung; Zhang, Rong; Jia, Weiping; Okada, Yukinori; Takahashi, Atsushi; Kubo, Michiaki; Tanaka, Toshihiro; Kamatani, Naoyuki; Matsuda, Koichi; Park, Taesung; Oh, Bermseok; Kimm, Kuchan; Kang, Daehee; Shin, Chol; Cho, Nam H; Kim, Hyung-Lae; Han, Bok-Ghee; Lee, Jong-Young; Cho, Yoon Shin

2011-09-11

To identify the genetic bases for nine metabolic traits, we conducted a meta-analysis combining Korean genome-wide association results from the KARE project (n = 8,842) and the HEXA shared control study (n = 3,703). We verified the associations of the loci selected from the discovery meta-analysis in the replication stage (30,395 individuals from the BioBank Japan genome-wide association study and individuals comprising the Health2 and Shanghai Jiao Tong University Diabetes cohorts). We identified ten genome-wide significant signals newly associated with traits from an overall meta-analysis. The most compelling associations involved 12q24.11 (near MYL2) and 12q24.13 (in C12orf51) for high-density lipoprotein cholesterol, 2p21 (near SIX2-SIX3) for fasting plasma glucose, 19q13.33 (in RPS11) and 6q22.33 (in RSPO3) for renal traits, and 12q24.11 (near MYL2), 12q24.13 (in C12orf51 and near OAS1), 4q31.22 (in ZNF827) and 7q11.23 (near TBL2-BCL7B) for hepatic traits. These findings highlight previously unknown biological pathways for metabolic traits investigated in this study.

Hydrogen-rich saline attenuates skin ischemia/reperfusion induced apoptosis via regulating Bax/Bcl-2 ratio and ASK-1/JNK pathway.

PubMed

Liu, Yun-Qi; Liu, Yi-Fang; Ma, Xue-Mei; Xiao, Yi-Ding; Wang, You-Bin; Zhang, Ming-Zi; Cheng, Ai-Xin; Wang, Ting-Ting; Li, Jia-La; Zhao, Peng-Xiang; Xie, Fei; Zhang, Xin

2015-07-01

Many pathways have been reported involving the effect of hydrogen-rich saline on protecting skin flap partial necrosis induced by the inflammation of ischemia/reperfusion injury. This study focused on the influence of hydrogen-rich saline treatment on apoptosis pathway of ASK-1/JNK and Bcl-2/Bax radio in I/R injury of skin flaps. Adult male Sprague-Dawley rats were divided into three groups. Group 1 was sham surgery group, Group 2 and 3 were ischemia/reperfusion surgery treated with physiological saline and hydrogen-rich saline respectively. Blood perfusion of flap was measured by Laser doppler flowmeters. Hematoxylin and eosin staining was used to observe morphological changes. Early apoptosis in skin flap was observed through TUNEL staining and presented as the percentage of TUNEL-positive cells of total cells. pASK-1, pJNK, Bcl-2 and Bax were examined by immunodetection. In addition Bcl-2, Bax and caspase-3 were detected by qPCR. Caspase-3 activity was also measured. Compared to the Group 2, tissues from the group 3 were observed with a high expression of Bcl-2 and a low expression of pASK-1, pJNK, and Bax, a larger survival area and a high level of blood perfusion. Hydrogen-rich saline ameliorated inflammatory infiltration and decreased cell apoptosis. The results indicate that hydrogen-rich saline could ameliorate ischemia/reperfusion injury and improve flap survival rate by inhibiting the apoptosis factor and, at the same time, promoting the expression of anti-apoptosis factor. Copyright © 2015. Published by Elsevier Ltd.

Therapeutic potential of GW501516 and the role of Peroxisome proliferator-activated receptor β/δ and B-cell lymphoma 6 in inflammatory signaling in human pancreatic cancer cells.

PubMed

Smith, Russell W; Coleman, Jeffrey D; Thompson, Jerry T; Vanden Heuvel, John P

2016-12-01

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a member of the nuclear receptor superfamily and a ligand-activated transcription factor that is involved in the regulation of the inflammatory response via activation of anti-inflammatory target genes and ligand-induced disassociation with the transcriptional repressor B-cell lymphoma 6 (BCL6). Chronic pancreatitis is considered to be a significant etiological factor for pancreatic cancer development, and a better understanding of the underlying mechanisms of the transition between inflammation and carcinogenesis would help further elucidate chemopreventative options. The aim of this study was to determine the role of PPARβ/δ and BCL6 in human pancreatic cancer of ductal origin, as well as the therapeutic potential of PPARβ/δ agonist, GW501516. Over-expression of PPARβ/δ inhibited basal and TNFα-induced Nfkb luciferase activity. GW501516-activated PPARβ/δ suppressed TNFα-induced Nfkb reporter activity. RNAi knockdown of Pparb attenuated the GW501516 effect on Nfkb luciferase, while knockdown of Bcl6 enhanced TNFα-induced Nfkb activity. PPARβ/δ activation induced expression of several anti-inflammatory genes in a dose-dependent manner, and GW501516 inhibited Mcp1 promoter-driven luciferase in a BCL6-dependent manner. Several pro-inflammatory genes were suppressed in a BCL6-dependent manner. Conditioned media from GW501516-treated pancreatic cancer cells suppressed pro-inflammatory expression in THP-1 macrophages as well as reduced invasiveness across a basement membrane. These results demonstrate that PPARβ/δ and BCL6 regulate anti-inflammatory signaling in human pancreatic cancer cells by inhibiting NFκB and pro-inflammatory gene expression, and via induction of anti-inflammatory target genes. Activation of PPARβ/δ may be a useful target in pancreatic cancer therapeutics.

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.

Apoptosis Modulation in the Immune System Reveals a Role of Neutrophils in Tissue Damage in a Murine Model of Chlamydial Genital Infection.

PubMed

Zortel, Tom; Schmitt-Graeff, Annette; Kirschnek, Susanne; Häcker, Georg

2018-05-05

Chlamydial infection frequently causes damage to the female genital tract. The precise mechanisms of chlamydial clearance and tissue damage are unknown, but studies suggest immunopathology with a particular role of neutrophils. The goal of this study was to understand the contribution of the immune system, in particular neutrophils. Using Chlamydia muridarum, we infected mice with a prolonged immune response due to expression of B-cell lymphoma 2 (Bcl-2) in hematopoietic cells (Bcl-2 mice), and mice where mature neutrophils are lacking due to the deletion of Myeloid cell leukemia 1 (Mcl-1) in myeloid cells (LysM-cre-mcl-1-flox mice; Mcl-1 mice). We monitored bacterial clearance, cellular infiltrate, and long-term tissue damage. Both mutant strains showed slightly delayed clearance of the acute infection. Bcl-2 mice had a strongly increased inflammatory infiltrate concerning almost all cell lineages. The infection of Bcl-2 mice caused increased tissue damage. The loss of neutrophils in Mcl-1 mice was associated with substantial quantitative and qualitative alterations of the inflammatory infiltrate. Mcl-1 mice had higher chlamydial burden and reduced tissue damage, including lower incidence of hydrosalpinx and less uterine dilation. Inhibition of apoptosis in the hematopoietic system increases inflammation and tissue damage. Neutrophils have broad functions, including a role in chlamydial clearance and in tissue destruction.

Naringin inhibits ovarian tumor growth by promoting apoptosis: An in vivo study.

PubMed

Cai, Liping; Wu, Heli; Tu, Chunhua; Wen, Xiaochun; Zhou, Bei

2018-07-01

The aim of the present study was to investigate the antitumor activities of naringin in ovarian cancer, and to assess the underlying mechanisms. Ovarian tumor cells were implanted into nude mice to produce ovarian tumors in vivo . The mice were divided into six groups: Control, low dose naringin [0.5 mg/kg, intraperitoneal (i.p.)], middle dose naringin (1 mg/kg, i.p.), high dose naringin (2 mg/kg, i.p.), positive control (cisplatin, 2 mg/kg, i.p.) and a combination of cisplatin and naringin (both 2 mg/kg). Following administration of naringin and/or cisplatin, the tumor size and weight were measured. Apoptosis of tumor cells was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis-associated gene expression was detected using reverse transcription-polymerase chain reaction and immunohistochemistry. In the range of 0.5-2 mg/kg, naringin dose-dependently inhibited tumor growth, as demonstrated by a decrease in tumor size and weight. Naringin promoted apoptosis of the ovarian tumor cells. Additionally, naringin reduced the expression of B-cell lymphoma (Bcl)-2, Bcl-extra large (Bcl-xL), cyclin D1, c-Myc and survivin, while it increased the expression of caspase-3 and caspase-7. The data demonstrated that naringin inhibited ovarian tumor growth in vivo . Its mechanisms may be associated with caspase-7-, caspase-3-, Bcl-2- and Bcl-xL-mediated apoptosis. Nevertheless, the clinical application of naringin in the treatment of ovarian cancer requires further study.

Effector mechanism of magnolol-induced apoptosis in human lung squamous carcinoma CH27 cells

PubMed Central

Yang, Shu-Er; Hsieh, Ming-Tsuen; Tsai, Tung-Hu; Hsu, Shih-Lan

2003-01-01

Magnolol, an active component isolated from the root and stem bark of Magnolia officinalis, has been reported to exhibit antitumour effects, but little is known about its molecular mechanisms of action. Magnolol inhibited proliferation of human lung squamous carcinoma CH27 cells at low concentrations (10–40 μM), and induced apoptosis at high concentrations (80–100 μM). Treatment with 80 μM magnolol significantly increased the expression of Bad and Bcl-XS proteins, whereas it decreased the expression of Bcl-XL. Overexpression of Bcl-2 protected CH27 cells against magnolol-triggered apoptosis. Magnolol treatment resulted in accumulation of cytosolic cytochrome c and activation of caspase-9 and downstream caspases (caspase-3 and -6). Pretreatment with z-VAD-fmk markedly inhibited magnolol-induced cell death, but did not prevent cytosolic cytochrome c accumulation. Magnolol induced a modest and persistent JNK activation and ERK inactivation in CH27 cells without evident changes in the protein levels. The responsiveness of JNK and ERK to magnolol suggests the involvement of these kinases in the initiation of the apoptosis process. These results indicate that regulation of the Bcl-2 family, accumulation of cytosolic cytochrome c, and activation of caspase-9 and caspase-3 may be the effector mechanisms of magnolol-induced apoptosis. PMID:12522090

A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

PubMed Central

Yang, Xiao-Feng; Weber, Georg F.

2014-01-01

Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

[Successful treatment of a patient with two hematologic tumors: double-hit lymphoma and acute myelomonoblastic leukemia].

PubMed

Lukina, A E; Bariakh, E A; Kravchenko, S K; Nareĭko, M V; Kuz'mina, L A; Parovichnikova, E N; Obukhova, T N; Kovrigina, A M; Magomedova, A U

2014-01-01

Double-hit (DH) lymphoma, an extremely aggressive variant of B-cell lymphoma, is accompanied by chromosomal abnormalities leading to the activation of a few oncogenes, one of which is the c-MYC gene in conjunction with BCL2 or BCL6 gene rearrangements. There are most common cases of MYC/8q24 and BCL2/18q21 gene rearrangements (MYC/BCL-2 DH lymphoma). The tumor is characterized by an aggressive clinical course and a poor response to chemotherapy (CT). The median survival in patients with DH lymphomas varies from 4.5 to 18 months. Such patients are generally resistant to CHOP-21 and R-CHOP-21 therapy regimens. For the treatment of patients with DH lymphoma, the Hematology Research Center, Ministry of Health of the Russian Federation, chose an original BL-M-04 polychemotherapy (PCT) protocol in combination with rituximab, followed by autologous stem cell transplantation (allo-SCT). The paper describes the experience in successfully treating a patient with two hematologic tumors: 1) MYC/BCL-2 DH lymphoma with high-dose PCT cycles, followed by allo-SCT, and 2) a metachronously developed second tumor (acute myelomonoblastic leukemia (AMML)) with CT cycles, followed by auto-SCT. The incidence of tumors induced by the previous high-dose CT for aggressive lymphomas for 10 years is 0.7 to 10%. As a rule, the development of secondary AMML is preceded by a history of myelodysplastic syndrome (MDS); characteristic chromosomal abnormalities (deletions of the long arm of chromosomes 5 and 7) are detectable. In this case, the follow-up was 3 months before the development of AMML, during this period the patient was not found to have laboratory signs of MDS (anemia, thrombocytopenia) or chromosomal abnormalities associated with secondary MDS/AML. The presence of a leukemic stem cell is associated with the occurrence and development of hemoblastosis; that of the similar cell populations that may cause B-cell lymphomas remains uncertain. The described case may have defect in a hematopoietic stem cell that gives rise to both germs of hematopoiesis, as well as complete donor chimerism of bone marrow hematopoiesis, which gives hope to long-term remission in both DH lymphoma and AMML.

Method of separating boron isotopes

DOEpatents

Jensen, R.J.; Thorne, J.M.; Cluff, C.L.

1981-01-23

A method of boron isotope enrichment involving the isotope preferential photolysis of (2-chloroethenyl)-dichloroborane as the feed material. The photolysis can readily by achieved with CO/sub 2/ laser radiation and using fluences significantly below those required to dissociate BCl/sub 3/.

Method of separating boron isotopes

DOEpatents

Jensen, Reed J.; Thorne, James M.; Cluff, Coran L.; Hayes, John K.

1984-01-01

A method of boron isotope enrichment involving the isotope preferential photolysis of (2-chloroethenyl)dichloroborane as the feed material. The photolysis can readily be achieved with CO.sub.2 laser radiation and using fluences significantly below those required to dissociate BCl.sub.3.

Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

PubMed Central

2013-01-01

Background Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure. Methods Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM. Results The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure. Conclusions These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD. PMID:23497247

Genistein induces apoptosis by the inactivation of the IGF-1R/p-Akt signaling pathway in MCF-7 human breast cancer cells.

PubMed

Chen, Jun; Duan, Yuxin; Zhang, Xing; Ye, Yu; Ge, Bo; Chen, Jian

2015-03-01

Genistein is an estrogenic soy-derived compound belonging to the isoflavone class and shows anti-cancer effects. However, the specific cell apoptosis mechanisms of genistein have not been fully understood. In this study, we investigated the specific cell apoptosis mechanisms of genistein and the potential involvement of the IGF1R-Akt-Bcl-2 and Bax-mediated pathways in human breast cancer cells in vitro. MCF-7 human breast cancer cells were treated with various concentrations of genistein, and cell proliferation was evaluated by the MTT assay. Morphological changes in treated cells were examined by Hoechst 33258 staining, and treated cells were examined by flow cytometry. The levels of IGF-1R, p-Akt, Bcl-2, and Bax protein expression and Bcl-2 and Bax mRNA expression were evaluated by western blot and RT-PCR, respectively. Genistein inhibited the proliferation of MCF-7 cells and induced cell apoptosis, as determined by Hoechst staining and flow cytometry analysis. Furthermore, genistein induced the inactivation of IGF-1R and p-Akt and downregulated the Bcl-2/Bax protein ratio. These results suggest that genistein inhibited cell proliferation by inactivating the IGF-1R-PI3 K/Akt pathway and decreasing the Bcl-2/Bax mRNA and protein expressions. Our findings help elucidate the mechanisms by which genistein may contribute to the prevention of breast cancer carcinogenesis.

Williamsport Area Community College Long Range Planning: The Long Range Plan, Update 1987.

ERIC Educational Resources Information Center

Williamsport Area Community Coll., PA.

This update to Williamsport Area Community College's (WACC's) 1984-89 long-range plan offers a status report on each of the plan's 78 objectives, reassigns responsibility for specific objectives to make the plan responsive to the current organizational structure of the college, and offers 11 new objectives for the 1986-87 academic year. After…

Anti-apoptotic A1 is not essential for lymphoma development in Eµ-Myc mice but helps sustain transplanted Eµ-Myc tumour cells.

PubMed

Mensink, Mark; Anstee, Natasha S; Robati, Mikara; Schenk, Robyn L; Herold, Marco J; Cory, Suzanne; Vandenberg, Cassandra J

2018-03-01

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-X L , but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a -/- A1-b fl/fl A1-c -/- Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

Nrf2/p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis.

PubMed

Son, Young-Ok; Pratheeshkumar, Poyil; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Zhang, Zhuo; Shi, Xianglin

2014-10-10

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nrf2/p62 Signaling in Apoptosis Resistance and Its Role in Cadmium-induced Carcinogenesis*

PubMed Central

Son, Young-Ok; Pratheeshkumar, Poyil; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Zhang, Zhuo; Shi, Xianglin

2014-01-01

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis. PMID:25157103

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

Prognostic significance of MYC, BCL2, and BCL6 rearrangements in patients with diffuse large B-cell lymphoma treated with cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab.

PubMed

Akyurek, Nalan; Uner, Aysegul; Benekli, Mustafa; Barista, Ibrahim

2012-09-01

Diffuse large B-cell lymphomas (DLBCLs) are a biologically heterogeneous group in which various gene alterations have been reported. The aim of this study was to investigate the frequency and prognostic impact of BCL2, BCL6, and MYC rearrangements in cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab (R-CHOP)-treated DLBCL cases. Tissue microarrays were constructed from 239 cases of DLBCL, and the expressions of CD10, BCL6, MUM1/IRF4, and BCL2 were evaluated by immunohistochemistry. MYC, BCL2, and BCL6 rearrangements were investigated by interphase fluorescence in situ hybridization on tissue microarrays. Survival analysis was constructed from 145 R-CHOP-treated patients. MYC, BCL2, and BCL6 rearrangements were detected in 14 (6%), 36 (15%), and 69 (29%) of 239 DLBCL patients. Double or triple rearrangements were detected in 7 (3%) of 239 DLBCL cases. Of these, 4 had BCL2 and MYC, 2 had BCL6 and MYC, and 1 had BCL2, BCL6, and MYC rearrangements. The prognosis of these cases was extremely poor, with a median survival of 9 months. MYC rearrangement was associated with significantly worse overall survival (P = .01), especially for the cases with GC phenotype (P = .009). BCL6 rearrangement also predicted significantly shorter overall survival (P = .04), especially for the non-GC phenotype (P = .03). BCL2 rearrangement had no prognostic impact on outcome. International Prognostic Index (P = .004) and MYC rearrangement (P = .009) were independent poor prognostic factors. Analysis of MYC gene rearrangement along with BCL2 and BCL6 is critical in identifying high-risk patients with poor prognosis. Copyright © 2011 American Cancer Society.

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

Assessment of Bax and Bcl-2 Immunoexpression in Patients with Oral Lichen Planus and Oral Squamous Cell Carcinoma

PubMed Central

Nafarzadeh, Shima; Jafari, Sina; Bijani, Ali

2013-01-01

Lichen planus (LP) is a chronic inflammatory disease of probable immune-based etiology. The pathogenesis of LP is unclear, but apoptotic changes in epidermal (epithelial) cells have been reported. Destruction of the basal cell layer is observed and many changes in cell proliferation, cell repair and cell death occur in the injured mucosal epithelium. The aim of this study was to evaluate and compare the expression of bax and bcl-2 in oral lichen planus (OLP), well differentiated oral squamous cell carcinoma (WOSCC) and normal mucosa. Sixty one paraffin-embedded biopsy including 11 cases of WOSCC, 30 cases of OLP (n=15 erosive OLP [OLP-E], n=15 reticular OLP [OLP-R]) and 20 normal mucosa were entered in our research. We used immunohistochemistry staining method for assessing bax and bcl-2 expression in epithelial layers. The percentage of stained cells was estimated in 5 randomized microscopic fields and classified as (-): 0%, (+) :< 10%, (++): 10-25%, (+++): 26-50%, (++++): > 50% positive cells. The data were analyzed with Mann-Whitney, Chi Square, and Kruskal-Wallis tests. Significant differences in bax expression were observed among OLP, WOSCC compared to normal mucosa (P=0.008). No significant difference in bax expression between OLP-E and OLP-R compared to WOSCC was seen (P>0.05). Bcl-2 was negative for all OLP and normal mucosa samples, and weak positivity was observed in WOSCC samples. According to the findings of our study, it may be possible to correlate the difference of bax and bcl-2 expression levels among the mentioned lesions to the malignant potential of OLP. PMID:24551804

A Systematic Review of Known Mechanisms of Hydroxyurea-induced Foetal Haemoglobin for Treatment of Sickle Cell Disease

PubMed Central

Pule, Gift D.; Mowla, Shaheen; Novitzky, Nicolas; Wiysonge, Charles S.; Wonkam, Ambroise

2016-01-01

Aims To report on molecular mechanisms of foetal haemoglobin (HbF) induction by hydroxyurea (HU) for the treatment of Sickle Cell Disease (SCD). Study Design Systematic review. Results Studies have provided consistent associations between genomic variations in HbF-promoting loci and variable HbF level in response to HU. Numerous signal transduction pathways have been implicated, through the identification of key genomic variants in BCL11A, HBS1L-MYB, SAR1 or XmnI polymorphism that predispose the response to the treatment, and signal transduction pathways, that modulate γ-globin expression (cAMP/cGMP; Giα/JNK/Jun; methylation and microRNA). Three main molecular pathways have been reported: 1) Epigenetic modifications, transcriptional events and signalling pathways involved in HU-mediated response, 2) Signalling pathways involving HU-mediated response and 3) Post-transcriptional pathways (regulation by microRNAs). Conclusions The complete picture of HU-mediated mechanisms of HbF production in SCD remains elusive. Research on post-transcriptional mechanisms could lead to therapeutic targets that may minimize alterations to the cellular transcriptome. PMID:26327494

A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease.

PubMed

Pule, Gift D; Mowla, Shaheen; Novitzky, Nicolas; Wiysonge, Charles S; Wonkam, Ambroise

2015-10-01

To report on molecular mechanisms of fetal hemoglobin (HbF) induction by hydroxyurea (HU) for the treatment of sickle cell disease. Systematic review. Studies have provided consistent associations between genomic variations in HbF-promoting loci and variable HbF level in response to HU. Numerous signal transduction pathways have been implicated, through the identification of key genomic variants in BCL11A, HBS1L-MYB, SAR1 or XmnI polymorphism that predispose the response to the treatment, and signal transduction pathways that modulate γ-globin expression (cAMP/cGMP; Giα/c-Jun N-terminal kinase/Jun; methylation and miRNA). Three main molecular pathways have been reported: i) Epigenetic modifications, transcriptional events and signaling pathways involved in HU-mediated response, ii) Signaling pathways involving HU-mediated response and iii) Post-transcriptional pathways (regulation by miRNAs). The complete picture of HU-mediated mechanisms of HbF production in Sickle Cell Disease remains elusive. Research on post-transcriptional mechanisms could lead to therapeutic targets that may minimize alterations to the cellular transcriptome.

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.

Transformation of Follicular Lymphoma to a High-Grade B-Cell Lymphoma With MYC and BCL2 Translocations and Overlapping Features of Burkitt Lymphoma and Acute Lymphoblastic Leukemia: A Case Report and Literature Review.

PubMed

Bischin, Alina M; Dorer, Russell; Aboulafia, David M

2017-01-01

Most commonly, histologic transformation (HT) from follicular lymphoma (FL) manifests as a diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). Less frequently, HT may result in a high-grade B-cell lymphoma (HGBL) with MYC and B-cell lymphoma protein 2 (BCL2) and/or BCL6 gene rearrangements, also known as "double-hit" or "triple-hit" lymphomas. In the 2016 revision of the World Health Organization (WHO) classification of lymphoid neoplasms, the category B-cell lymphoma, unclassifiable was eliminated due to its vague criteria and limiting diagnostic benefit. Instead, the WHO introduced the HGBL category, characterized by MYC and BCL2 and/or BCL6 rearrangements. Cases that present as an intermediate phenotype of DLBCL and Burkitt lymphoma (BL) will fall within this HGBL category. Very rarely, HT results in both the intermediate DLBCL and BL phenotypes and exhibits lymphoblastic features, in which case the WHO recommends that this morphologic appearance should be noted. In comparison with de novo patients with DLBCL, NOS, those with MYC and BCL2 and/or BCL6 gene rearrangements have a worse prognosis. A 63-year-old woman presented with left neck adenopathy. Laboratory assessments, including complete blood count, complete metabolic panel, serum lactate dehydrogenase, and β 2 -microglobulin, were all normal. A whole-body computerized tomographic (CT) scan revealed diffuse adenopathy above and below the diaphragm. An excisional node biopsy showed grade 3A nodular FL. The Ki67 labeling index was 40% to 50%. A bone marrow biopsy showed a small focus of paratrabecular CD20+ lymphoid aggregates. She received 6 cycles of bendamustine (90 mg/m2 on days +1 and +2) and rituximab (375 mg/m2 on day +2), with each cycle delivered every 4 weeks. A follow-up CT scan at completion of therapy showed a partial response with resolution of axillary adenopathy and a dramatic shrinkage of the large retroperitoneal nodes. After 18 months, she had crampy abdominal pain in the absence of B symptoms. Positron emission tomography with 2-deoxy-2-[fluorine-18] fluoro-d-glucose integrated with CT (18F-FDG PET/CT) scan showed widespread adenopathy, diffuse splenic involvement, and substantial marrow involvement. Biopsy of a 2.4-cm right axillary node (SUVmax of 16.1) showed involvement by grade 3A FL with a predominant nodular pattern of growth. A bone marrow biopsy once again showed only a small focus of FL. She received idelalisib (150 mg twice daily) and rituximab (375 mg/m2, monthly) beginning May 2015. After 4 cycles, a repeat CT scan showed a complete radiographic response. Idelalisib was subsequently held while she received corticosteroids for immune-mediated colitis. A month later, she restarted idelalisib with a 50% dose reduction. After 2 weeks, she returned to clinic complaining of bilateral hip and low lumbar discomfort but no B symptoms. A restaging 18F-FDG PET/CT in January 2016 showed dramatic marrow uptake. A bone marrow aspirate showed sheets of tumor cells representing a spectrum from intermediate-sized cells with lymphoblastic features to very large atypical cells with multiple nucleoli. Two distinct histologies were present; one remained consistent with the patient's known FL with a predominant nodular pattern and the other consistent with HT (the large atypical cells expressed PAX5, CD10, BCL2, and c-MYC and were negative for CD20, MPO, CD34, CD30, and BCL6). Focal areas showed faint, heterogeneous expression of terminal deoxynucleotidyl transferase best seen on the clot section. Ki67 proliferation index was high (4+/4). Fluorescence in situ hybridization analysis showed 2 populations with MYC amplification and/or rearrangement and no evidence of BCL6 rearrangement; a karyotype analysis showed a complex abnormal female karyotype with t(14;18) and multiple structural and numerical abnormalities. She started dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin with concomitant prophylactic intrathecal methotrexate and cytarabine. She had but a short-lived response before dying in hospice from progressive lymphoma. Whether idelalisib could provide a microenvironment for selection of more aggressive clones needs to be addressed. Our patient's clinical course is confounded by the incorporation of idelalisib while being further complicated by the complexity of HT and the mechanisms in which first-line chemotherapy regimens affect double-hit lymphoma.

Translating neurotrophic and cellular plasticity: from pathophysiology to improved therapeutics for bipolar disorder.

PubMed

Soeiro-de-Souza, M G; Dias, V V; Figueira, M L; Forlenza, O V; Gattaz, W F; Zarate, C A; Machado-Vieira, R

2012-11-01

Bipolar disorder (BD) likely involves, at a molecular and cellular level, dysfunctions of critical neurotrophic, cellular plasticity and resilience pathways and neuroprotective processes. Therapeutic properties of mood stabilizers are presumed to result from a restoration of the function of these altered pathways and processes through a wide range of biochemical and molecular effects. We aimed to review the altered pathways and processes implicated in BD, such as neurotrophic factors, mitogen-activated protein kinases, Bcl-2, phosphoinositol signaling, intracellular calcium and glycogen synthase kinase-3. We undertook a literature search of recent relevant journal articles, book chapter and reviews on neurodegeneration and neuroprotection in BD. Search words entered were 'brain-derived neurotrophic factor,''Bcl-2,''mitogen-activated protein kinases,''neuroprotection,''calcium,''bipolar disorder,''mania,' and 'depression.' The most consistent and replicated findings in the pathophysiology of BD may be classified as follows: i) calcium dysregulation, ii) mitochondrial/endoplasmic reticulum dysfunction, iii) glial and neuronal death/atrophy and iv) loss of neurotrophic/plasticity effects in brain areas critically involved in mood regulation. In addition, the evidence supports that treatment with mood stabilizers; in particular, lithium restores these pathophysiological changes. Bipolar disorder is associated with impairments in neurotrophic, cellular plasticity and resilience pathways as well as in neuroprotective processes. The evidence supports that treatment with mood stabilizers, in particular lithium, restores these pathophysiological changes. Studies that attempt to prevent (intervene before the onset of the molecular and cellular changes), treat (minimize severity of these deficits over time), and rectify (reverse molecular and cellular deficits) are promising therapeutic strategies for developing improved treatments for bipolar disorder. © 2012 John Wiley & Sons A/S.

PD1 and PDL1 expression in primary central nervous system diffuse large B-cell lymphoma are frequent and expression of PD1 predicts poor survival.

PubMed

Four, Marion; Cacheux, Valère; Tempier, Ariane; Platero, Dolorès; Fabbro, Michel; Marin, Grégory; Leventoux, Nicolas; Rigau, Valérie; Costes-Martineau, Valérie; Szablewski, Vanessa

2017-12-01

Primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) is a rare and aggressive type of diffuse large B-cell lymphoma (DLBCL) whit poorly understood pathogenesis. Finding biomarkers associated with patient survival may be important for understanding its physiopathology and to develop new therapeutic approaches. We investigated 32 PCNS-DLBCL from immunocompetent patients for BCL2, CMYC, LMO2, and P53 expression and for cytogenetic aberrations of BCL2, BCL6, and MYC genes, all known for their prognostic value in systemic DLBCL (s-DLBCL). We analyzed PD1 and PDL1 protein expression in both tumor infiltrating lymphocytes (TILs) and tumor cells. Finally, we searched for correlation between biological data and clinical course. The PCNS-DLBCL expressed BCL2, CMYC, LMO2, and P53 at similar frequency than s-DLBCL but without significant prognostic on survival. None cases harbored aberrations involving BCL2 and MYC gene whereas BCL6 abnormalities were present in 20.7% of cases but without value on survival. Expression of PD1 in TILs and PDL1 in tumor cells was observed at higher rates than in s-DLBCL (58% and 37%, respectively). The PD1 expression in TILs correlated with PDL1 expression in tumor cells (P = .001). Presence of PD1 positive TILs was associated with poorer overall survival (P = .011). Patients with PDL1 overexpression tended to better response to chemotherapy (P = .23). In conclusion PCNS-DLBCL pathogenesis differs from s-DLBCL without prognostic value of the phenotypic and cytogenetic parameters known for their pejorative impact in the latter. The PD1/PDL1 pathway plays a strong role in PCNS-DLBCL and represents an attractive target for this aggressive lymphoma. Copyright © 2016 John Wiley & Sons, Ltd.

Translating neurotrophic and cellular plasticity: from pathophysiology to improved therapeutics for bipolar disorder

PubMed Central

Soeiro-de-Souza, M. G.; Dias, V. V.; Figueira, M. L.; Forlenza, O. V.; Gattaz, W. F.; Zarate, C. A.; Machado-Vieira, R.

2014-01-01

Objective Bipolar disorder (BD) likely involves, at a molecular and cellular level, dysfunctions of critical neurotrophic, cellular plasticity and resilience pathways and neuroprotective processes. Therapeutic properties of mood stabilizers are presumed to result from a restoration of the function of these altered pathways and processes through a wide range of biochemical and molecular effects. We aimed to review the altered pathways and processes implicated in BD, such as neurotrophic factors, mitogen-activated protein kinases, Bcl-2, phosphoinositol signaling, intracellular calcium and glycogen synthase kinase-3. Methods We undertook a literature search of recent relevant journal articles, book chapter and reviews on neurodegeneration and neuroprotection in BD. Search words entered were ‘brain-derived neurotrophic factor,’ ‘Bcl-2,’ ‘mitogen-activated protein kinases,’ ‘neuroprotection,’ ‘calcium,’ ‘bipolar disorder,’ ‘mania,’ and ‘depression.’ Results The most consistent and replicated findings in the pathophysiology of BD may be classified as follows: i) calcium dysregulation, ii) mitochondrial/endoplasmic reticulum dysfunction, iii) glial and neuronal death/atrophy and iv) loss of neurotrophic/plasticity effects in brain areas critically involved in mood regulation. In addition, the evidence supports that treatment with mood stabilizers; in particular, lithium restores these pathophysiological changes. Conclusion Bipolar disorder is associated with impairments in neurotrophic, cellular plasticity and resilience pathways as well as in neuroprotective processes. The evidence supports that treatment with mood stabilizers, in particular lithium, restores these pathophysiological changes. Studies that attempt to prevent (intervene before the onset of the molecular and cellular changes), treat (minimize severity of these deficits over time), and rectify (reverse molecular and cellular deficits) are promising therapeutic strategies for developing improved treatments for bipolar disorder. PMID:22676371

MiR-34a, a promising novel biomarker for benzene toxicity, is involved in cell apoptosis triggered by 1,4-benzoquinone through targeting Bcl-2.

PubMed

Chen, Yujiao; Sun, Pengling; Guo, Xiaoli; Gao, Ai

2017-02-01

Exposure to benzene is inevitable, and concerns regarding the adverse health effects of benzene have been raised. Most investigators found that benzene exposure induced hematotoxicity. In this regard, Our study aimed to explore a novel potential biomarker of adverse health effects following benzene exposure and the toxic mechanisms of benzene metabolites in vitro. This study consisted of 314 benzene-exposed workers and 288 control workers, an air benzene concentration of who were 2.64 ± 1.60 mg/m 3 and 0.05 ± 0.01 mg/m 3 , respectively. In this population-based study, miR-34a expression was elevated in benzene-exposed workers. The correlation of miR-34a with the airborne benzene concentration, S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t, t-MA), all of which reflect benzene exposure, was found. Correlation analysis indicated that miR-34a was associated with peripheral blood count, alanine transaminase (ALT) and oxidative stress. Furthermore, multivariate analysis demonstrated that miR-34a expression was strongly associated with white blood cell count (structure loadings = 0.952). In population-based study, miR-34a had the largest contribution to altered peripheral blood counts, which reflect benzene-induced hematotoxicity. The role of miR-34a in benzene toxicity was assessed using lentiviral vector transfection. Results revealed that 1,4-benzoquinone induced abnormal cell apoptosis and simultaneously upregulated miR-34a accompanied with decreased Bcl-2. Finally, inhibition of miR-34a elevated Bcl-2 and decreased 1,4-benzoquinone-induced apoptosis. In conclusion, miR-34a was observed to be involved in benzene-induced hematotoxicity by targeting Bcl-2 and could be regarded as a potential novel biomarker for benzene toxicity. Copyright © 2016 Elsevier Ltd. 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

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

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263.

PubMed

Curry, Merril; Roberts-Thomson, Sarah J; Monteith, Gregory R

2016-09-30

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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.

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells.

PubMed

Ho, Cheong-Yip; Kim, Chi-Fai; Leung, Kwok-Nam; Fung, Kwok-Pui; Tse, Tak-Fu; Chan, Helen; Lau, Clara Bik-San

2005-06-01

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.

Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.

PubMed

Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

2013-05-10

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

PubMed

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Markers of apoptosis and proliferation related gene products as predictors of treatment outcome in childhood acute lymphoblastic leukemia.

PubMed

Hafez, Mohammad; Al-Tonbary, Youssef; El-Bayoumi, Mohammed A; Hatem, Nadia; Hawas, Samia; Mansour, Ahmed; Marzouk, Iman; Hafez, Mona M; Yahia, Sohier; Farahat, Nahla

2007-06-01

The aim of the study is to characterize markers of apoptosis in children with acute lymphoblastic leukemia (ALL) in relation to treatment outcome of the disease. The study was performed on 34 children with ALL and 39 healthy children as a control group. Apoptosis was assessed by cell morphology; DNA fragmentation; ELISA and RT-PCR for CD95, CD95L, BcL-2 and nuclear factor-kappa B (NF-kappaB); and flow cytometry for CD95, CD40, CD49d and CD11a. Apoptosis was significantly lower in patients than controls. Apoptosis detected by CD95 ligand was significantly lower in cases with no remission after treatment than those who achieved remission. Anti-apoptotic factors: CD40, BcL-2, and NF-kappaB were all found to be higher in cases than controls and in cases with no remission than those achieved remission. CD49d was significantly lower in cases than controls, and significantly lower in cases with who did not achieve remission. CD11a levels were similar in the various groups. Delayed apoptosis of ALL cells is genetically controlled either directly or indirectly by a network of oncogenes and tumor suppressor genes. CD40 appeared to stimulate both T and B lineage and is considered the most potent influencer and predictor of resistance to therapy. Inhibitors for the activity of CD40, Bcl-2 and NF-kappaB as well as stimulants to CD95 could have a potential therapeutic benefit.

Dexamethasone treatment promotes Bcl-2 dependence in multiple myeloma resulting in sensitivity to venetoclax.

PubMed

Matulis, S M; Gupta, V A; Nooka, A K; Hollen, H V; Kaufman, J L; Lonial, S; Boise, L H

2016-05-01

Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. The results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in four of the five cell lines tested. The most striking results were seen with dexamethasone (Dex). Co-treatment of human myeloma cell lines and primary patient samples, with Dex and venetoclax, significantly increased cell death over venetoclax alone in four of the five cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of Dex. This results in alterations in Bim binding to anti-apoptotic proteins. Dex shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim-binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with Dex could be an effective therapy for a broader range of patients than would be predicted by single-agent activity.

Dexamethasone treatment promotes Bcl-2-dependence in multiple myeloma resulting in sensitivity to Venetoclax

PubMed Central

Matulis, Shannon M.; Gupta, Vikas A.; Nooka, Ajay K.; Von Hollen, Hayley; Kaufman, Jonathan L.; Lonial, Sagar; Boise, Lawrence H.

2015-01-01

Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. Results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in 4 of the 5 cell lines tested. The most striking results were seen with dexamethasone. Co-treatment of human myeloma cell lines and primary patient samples, with dexamethasone and venetoclax significantly increased cell death over venetoclax alone in 4 of the 5 cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of dexamethasone. This results in alterations in Bim binding to anti-apoptotic proteins. Dexamethasone shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with dexamethasone could be an effective therapy for a broader range of patients than would be predicted by single agent activity. PMID:26707935

Protein phosphatases 2A as well as reactive oxygen species involved in tributyltin-induced apoptosis in mouse livers.

PubMed

Zhang, Yali; Chen, Yonggang; Sun, Lijun; Liang, Jing; Guo, Zonglou; Xu, Lihong

2014-02-01

Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce caspase-3-dependent apoptosis in human amniotic cells through protein phosphatase 2A (PP2A) inhibition and consequent JNK activation. This in vivo study was undertaken to further verify the results derived from our previous in vitro study. Mice were orally dosed with 0, 10, 20, and 60 mg/kg of body weight TBT, and levels of PP2A, reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK), Bax/Bcl-2, and caspase-3 were detected in the mouse livers. Apoptosis was also evaluated using the TUNEL assay. The results showed that PP2A activity was inhibited, ROS levels were elevated, and MAPKs including ERK, JNK, and p38 were activated in mouse livers treated with the highest dose of TBT. Additionally, the ratio of Bax/Bcl-2 was increased, caspase-3 was activated, and apoptosis in mouse livers could be detected in the highest dose group. Therefore, a possible signaling pathway in TBT-induced apoptosis in mouse livers involves PP2A inhibition and ROS elevation serving a pivotal function as upstream activators of MAPKs; activation of MAPKs in turn leads to an increase in the Bax/Bcl-2 ratio, ultimately leading to the activation of caspase-3. The results give a comprehensive and novel description of the mechanism of TBT-induced toxicity. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

Trastuzumab down-regulates Bcl-2 expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene--amplified breast cancer cells.

PubMed

Milella, Michele; Trisciuoglio, Daniela; Bruno, Tiziana; Ciuffreda, Ludovica; Mottolese, Marcella; Cianciulli, Anna; Cognetti, Francesco; Zangemeister-Wittke, Uwe; Del Bufalo, Donatella; Zupi, Gabriella

2004-11-15

To investigate the possible existence of an antiapoptotic cross-talk between HER-2 and antiapoptotic Bcl-2 family members. Bcl-2 and Bcl-XL expression and apoptosis induction were analyzed in HER-2 gene-amplified (BT474) and nonamplified (ZR 75-1) breast cancer cell lines exposed to trastuzumab, alone or in combination with either Bcl-2/Bcl-XL bispecific antisense oligonucleotides (AS-4625) or the small-molecule Bcl-2 antagonist HA14-1. In addition to HER-2 and epidermal growth factor receptor, trastuzumab down-regulated Bcl-2, but not Bcl-XL, protein, and mRNA expression in BT474 cells. Interestingly, trastuzumab-induced down-regulation of HER-2 and Bcl-2 was also observed in three of five and two of three breast cancer patients undergoing trastuzumab treatment, respectively. Despite Bcl-2 down-regulation, however, trastuzumab only marginally increased the rate of apoptosis (7.3 +/- 3.5%). We therefore investigated whether a combination of AS-4625 and trastuzumab might increase proapoptotic efficiency. AS-4625 treatment of BT474 cells decreased both Bcl-2 and Bcl-XL expression, resulting in a 21 +/- 7% net apoptosis induction; the combination of AS-4625 followed by trastuzumab resulted in a significantly stronger induction of apoptosis (37 +/- 6%, P <0.01) that was not observed with the reverse treatment sequence (trastuzumab followed by AS-4625). Similar results were obtained with the Bcl-2 antagonist HA14-1; indeed, exposure of BT474 cells to HA14-1 followed by trastuzumab resulted in a striking proapoptotic synergism (combination index=0.58 +/- 0.18), as assessed by isobologram analysis. Altogether our findings suggest that combined targeting of HER-2 and Bcl-2 may represent a novel, rational approach to more effective breast cancer therapy.

[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.

Attenuation of warm ischemia-reperfusion injury in the liver by bucillamine through decreased neutrophil activation and Bax/Bcl-2 modulation.

PubMed

Junnarkar, Sameer P; Tapuria, Niteen; Mani, Alireza; Dijk, Sas; Fuller, Barry; Seifalian, Alexander M; Davidson, Brian R

2010-12-01

Liver transplantation and resection surgery involve a period of ischemia and reperfusion to the liver, which initiates an inflammatory cascade resulting in liver and remote organ injury. Bucillamine is a low molecular weight thiol antioxidant that is capable of rapidly entering cells. We hypothesized that bucillamine acts by replenishing glutathione levels, thus reducing neutrophil activation, modulating Bax/Bcl-2 expression, and subsequently, attenuating the effects of warm ischemia-reperfusion injury (IRI) in the liver. The effect of bucillamine was studied in a rat model of liver IRI with 45 min of partial (70%) liver ischemia and 3 h of reperfusion. Liver injury was assessed by measuring serum transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) and liver histology. Oxidative stress was quantified by measuring F(2) isoprostane and glutathione levels. Leukocyte adhesion was assessed by intravital microscopy, and inflammatory cytokine response was assessed by measuring serum cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels. Bax and Bcl-2 expression was measured by reverse transcription-polymerase chain reaction. The model produced significant liver injury with elevated transaminases and an acute inflammatory response. Bucillamine reduced the liver injury, as indicated by reduced AST (932 ± 200.8 vs 2072.5 ± 511.79, P < 0.05). Bucillamine reduced Bax expression, serum CINC-1 levels, and neutrophil adhesion, and upregulated Bcl-2. However, bucillamine did not affect tissue glutathione levels nor the levels of oxidative stress, as measured by plasma and hepatic F(2) isoprostane levels. Bucillamine reduces warm ischemia-reperfusion in the liver by inhibiting neutrophil activation and modulating Bax/Bcl-2 expression. © 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

Sensory Neuropathy Due to Loss of Bcl-w

PubMed Central

Courchesne, Stephanie L.; Karch, Christoph; Pazyra-Murphy, Maria F.; Segal, Rosalind A.

2010-01-01

Small fiber sensory neuropathy is a common disorder in which progressive degeneration of small diameter nociceptors causes decreased sensitivity to thermal stimuli and painful sensations in the extremities. In the majority of patients, the cause of small fiber sensory neuropathy is unknown, and treatment options are limited. Here, we show that Bcl-w (Bcl-2l2) is required for the viability of small fiber nociceptive sensory neurons. Bcl-w −/− mice demonstrate an adult-onset progressive decline in thermosensation and a decrease in nociceptor innervation of the epidermis. This denervation occurs without cell body loss, indicating that lack of Bcl-w results in a primary axonopathy. Consistent with this phenotype, we show that Bcl-w, in contrast to the closely related Bcl-2 and Bcl-xL, is enriched in axons of sensory neurons and that Bcl-w prevents the dying back of axons. Bcl-w −/− sensory neurons exhibit mitochondrial abnormalities, including alterations in axonal mitochondrial size, axonal mitochondrial membrane potential, and cellular ATP levels. Collectively, these data establish bcl-w −/− mice as an animal model of small fiber sensory neuropathy, and provide new insight regarding the role of bcl-w and of mitochondria in preventing axonal degeneration. PMID:21289171

Effect of fetal hypothyroidism on tolerance to ischemia-reperfusion injury in aged male rats: Role of nitric oxide.

PubMed

Jeddi, Sajad; Zaman, Jalal; Ghasemi, Asghar

2016-05-01

Aging is associated with increased prevalence of cardiovascular disease. Thyroid hormone deficiency during fetal life decreases myocardial tolerance to ischemia-reperfusion (IR) injury in later life. The long-term effects of fetal hypothyroidism (FH) on response to IR injury in aged rats have not been well documented. The aim of this study was therefore to compare the effect of FH on tolerance to IR injury in young and aged male rats and to determine contribution of iNOS (inducible nitric oxide synthase), Bax, and Bcl-2. Pregnant female rats were divided into two groups: The FH group received water containing 0.025% 6-propyl-2-thiouracil during gestation and the controls consumed tap water. Isolated perfused hearts from young (3 months) and aged (12 months) rats were subjected to IR. Hemodynamic parameters, infarct size, and heart NOx (nitrite+nitrate) levels were measured; in addition, mRNA expression of iNOS, Bax, and Bcl-2 and their protein levels in heart were measured. Recovery of post-ischemic LVDP and ±dp/dt were lower and infarct sizes were higher than controls in aged FH rats (68.38 ± 6.7% vs. 50.5 ± 1.7%; P < 0.05). Aged FH rats had higher heart NOx values than controls (74.3 ± 2.6 vs. 47.6 ± 2.5 μmol/L, P < 0.05). After IR, in FH rats, mRNA expression of iNOS and Bax were higher and Bcl-2 was lower in both the young (350 and 240% for iNOS and Bax, respectively and 51% for Bcl-2) and aged rats (504 and 567% for iNOS and Bax, respectively and 67% for Bcl-2). Compared to controls, in FH rats protein levels of iNOS (37% for young and 45% for aged rats) and Bax (94% for young and 118% for aged rats) were higher while for Bcl-2 (36% for young and 62% for aged rats) were lower. After IR, in FH rats, aminoguanidine, a selective iNOS inhibitor, decreased mRNA expression of iNOS and Bax and increased expression of Bcl-2 in both young (65% and 58% for iNOS and Bax, respectively and 152% for Bcl-2) and aged rats (76% and 64% for iNOS and Bax, respectively and 222% for Bcl-2). In addition, in the heart of FH rats, aminoguanidine decreased protein levels of iNOS (47% for young and 60% for aged rats) and Bax (57% for young and 80% for aged rats) and increased protein levels of Bcl-2 (124% for young and 180% for aged rats). In conclusion, thyroid hormone deficiency during fetal life decreases tolerance to IR injury in aged rats; this effect is at least in part, due to increased expression of iNOS and Bax-to-Bcl-2 ratio in the heart and is restored by iNOS inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

Long-term tolerance of airway silicone stent in patients with post-tuberculosis tracheobronchial stenosis.

PubMed

Verma, Akash; Um, Sang-Won; Koh, Won-Jung; Suh, Gee Young; Chung, Man Pyo; Kwon, O Jung; Kim, Hojoong

2012-01-01

Surgery is a well-recognized modality of treatment for benign tracheobronchial stenosis. However, in some benign disease groups, such as tuberculosis, sarcoidosis, war gas exposure, and inhalation burns, multiplicity of involvement or long length of stenosed segment heightens surgical challenge. We investigated the outcomes and long-term tolerability of the Natural stent (N-stent) in such patients with post-tuberculosis tracheobronchial stenosis. A retrospective review was done for 17 patients who underwent silicone stenting (N-stent) for post-tuberculosis tracheobronchial stenosis during January 2000-December 2003 but needed persistent stent placement and still require the stent. Significant increase in the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC), expressed as FEV1%, as well as in forced percentual vital capacity (FVC%) (Δ24% and Δ11%, respectively) in the short term and sustained increase in the FEV1% and FVC% (Δ26.5% and Δ16.5%, respectively) in the long term were noted at a median (range) interval of 1 (0.5-72) month and 72 (12-114) months, respectively, along with symptomatic relief. No procedure-related death occurred. Stent-related late complications included granulation tissue formation (76%), migration (70%), and mucostasis (17%). The median duration for which N-stents were tolerated was 7.9 (range, 3-11) years. N-stents are well-tolerated for a prolonged period. Stent-related complications occur, but are easily managed. These results might carry medical implications for those who have airway lesions difficult to correct surgically.

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

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.

Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model.

PubMed

Hollister, Kristin; Kusam, Saritha; Wu, Hao; Clegg, Ninah; Mondal, Arpita; Sawant, Deepali V; Dent, Alexander L

2013-10-01

The transcriptional repressor Bcl6 controls development of the follicular Th cell (T(FH)) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring T(FH) cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) T(FH) cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the T(FH) cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting T(FH) cell survival.

Mcl-1 and Bcl-xL Cooperatively Maintain Integrity of Hepatocytes in Developing and Adult Murine Liver

PubMed Central

Hikita, Hayato; Takehara, Tetsuo; Shimizu, Satoshi; Kodama, Takahiro; Li, Wei; Miyagi, Takuya; Hosui, Atsushi; Ishida, Hisashi; Ohkawa, Kazuyoshi; Kanto, Tatsuya; Hiramatsu, Naoki; Yin, Xiao-Ming; Hennighausen, Lothar; Tatsumi, Tomohide; Hayashi, Norio

2013-01-01

Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, especially in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1– deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL– deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL)-positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte-specific Bcl-xL– deficient mice. Although mcl-1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+ mcl-1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL– deficient or Mcl-1– deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+ AlbCre, bcl-xflox/+ mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner. PMID:19676108

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.

MitomiRs in human inflamm-aging: a hypothesis involving miR-181a, miR-34a and miR-146a.

PubMed

Rippo, Maria Rita; Olivieri, Fabiola; Monsurrò, Vladia; Prattichizzo, Francesco; Albertini, Maria Cristina; Procopio, Antonio Domenico

2014-08-01

Mitochondria are intimately involved in the aging process. The decline of autophagic clearance during aging affects the equilibrium between mitochondrial fusion and fission, leading to a build-up of dysfunctional mitochondria, oxidative stress, chronic low-grade inflammation, and increased apoptosis rates, the main hallmarks of aging. Current research suggests that a large number of microRNAs (miRs or miRNAs) are differentially expressed during cell aging. Other lines of evidence indicate that several miRs likely share in "inflamm-aging", an aging-related state characterized by systemic chronic inflammation that in turn provides a biological background favoring susceptibility to age-related diseases and disabilities. Interestingly, miRs can modulate mitochondrial activity, and a discrete miR set has recently been identified in mitochondria of different species and cell types (mitomiRs). Here we show that some mitomiRs (let7b, mir-146a, -133b, -106a, -19b, -20a, -34a, -181a and -221) are also among the miRs primarily involved in cell aging and in inflamm-aging. Of note, Ingenuity Pathway Analysis (IPA) of aging-related mitomiR targets has disclosed a number of resident mitochondrial proteins playing large roles in energy metabolism, mitochondrial transport and apoptosis. Among these, Bcl-2 family members--which are critically involved in maintaining mitochondrial integrity--may play a role in controlling mitochondrial function and dysfunction during cellular aging, also considering that Bcl-2, the master member of the family, is an anti-oxidant and anti-apoptotic factor and regulates mitochondrial fission/fusion and autophagy. This intriguing hypothesis is supported by several observations: i) in endothelial cells undergoing replicative senescence (HUVECs), a well-established model of cell senescence, miR-146a, miR-34a, and miR-181a are over-expressed whereas their target Bcl-2 is down-regulated; ii) IPA of the miR-146a, miR-34a and miR-181a network shows that they are closely linked to each other, to Bcl-2 and to mitochondria; and iii) miR-146a, miR-34a, and miR-181a are involved in important cell functions (growth, proliferation, death, survival, maintenance) and age-related diseases (cancer, skeletal and muscle disorders, neurological, cardiovascular and metabolic diseases). In conclusion several aging-related mitomiRs may play a direct role in controlling mitochondrial function by regulating mitochondrial protein expression. Their modulation could thus mediate the loss of mitochondrial integrity and function in aging cells, inducing or contributing to the inflammatory response and to age-related diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

IgV(H) and bcl6 somatic mutation analysis reveals the heterogeneity of cutaneous B-cell lymphoma, and indicates the presence of undisclosed local antigens.

PubMed

Franco, Renato; Camacho, Francisca I; Fernández-Vázquez, Amalia; Algara, Patrocinio; Rodríguez-Peralto, José L; De Rosa, Gaetano; Piris, Miguel A

2004-06-01

Our understanding of the ontology of B-cell lymphomas (BCL) has been improved by the study of mutational status of IgV(H) and bcl6 genes, but only a few cases of cutaneous BCL have been examined for this status. We analyzed IgV(H) and bcl6 somatic mutations in 10 cutaneous BCL, classified as follicular (three primary and one secondary), primary marginal zone (two cases), and diffuse large BCL (three primary and one secondary). We observed a lower rate (<2%) of IgV(H) mutation in all marginal zone lymphomas, and a preferential usage of V(H)2-70 (one primary follicular and two primary diffuse large BCL). Fewer than expected replacement mutations in framework regions (FR) were observed in three primary follicular lymphomas (FLs) and in all diffuse large BCL, indicating a negative antigen selection pressure. Ongoing mutations were observed in eight of 10 cases. Only two primary FLs and two diffuse large BCL showed bcl6 somatic mutation. These data support the heterogeneous nature of the different cutaneous BCL, and specifically the distinction between cutaneous follicular and marginal zone lymphomas. The biased usage of V(H)2-70, the low rate of replacement mutation in the FR, and the presence of ongoing mutation imply that local antigens could modulate the growth of primary cutaneous BCL.

[The effects of leptin on apoptosis of airway smooth muscle cells via the PI3K/Akt signaling pathway].

PubMed

Liu, Wen-jing; Zhu, Shu-yang; Chen, Yu-ling; Wu, Xia; Ni, Wen-jing; Chen, Yun-feng; Zhao, Ling

2012-12-01

To observe the effects of leptin on the expression of Akt, Pho-Akt, Bcl-2, Bax, caspase-3 and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms. ASMCs were derived from rat airway tissue and cultured in vitro. The cells were randomly divided into 5 groups including a control group, leptin at concentrations of 50, 100, 200 µg/L groups (group Lep50, Lep100, Lep200), and PI3K specific antagonist with Lep200 group. Then the cells of different groups were incubated for 24 h. An apoptosis detection kit was used for annexin V and PI staining. The expression of Akt, phosphorylation Akt, Bcl-2, Bax, caspase-3 were measured by Western blot. The apoptosis rates of ASMCs in group Lep50, Lep100 and Lep200 were (3.97 ± 0.39)%, (1.88 ± 0.72)% and (0.77 ± 0.11)%, respectively, all significantly lower than that in the control group (7.38 ± 0.49)% (F = 89.57, P < 0.05). Furthermore, the concentration of leptin was negatively related to the apoptosis rate (r = -0.711, P < 0.05). The apoptosis rates of PI3K specific antagonist with Lep200 group (3.29 ± 0.36)% was higher than that of group Lep200 (0.77 ± 0.11)% (F = 89.57, P < 0.01). After the intervention of leptin, the expression of Bcl-2 was upregulated and positively correlated with leptin concentration (r = 0.939, P < 0.05); Bax was downregulated and negatively related to the leptin concentration (r = -0.908, P < 0.05); while the Bcl-2/Bax ratio was raised after leptin treatment (F = 20.56, P < 0.05). Leptin inhibited the activation of caspase-3 in the negative way. (r = -0.961, P < 0.05). The results also showed that leptin significantly increased phosphorylation of Akt that positively related to leptin concentration (r = 0.958, P < 0.05). Compared with group Lep200, the expression of Pho-Akt and Bcl-2 in PI3K specific antagonist with Lep200 group were downregulated (F = 32.93, 19.48, respectively, P < 0.05), while the expression of Bax and caspase-3 was increased (F = 10.10, 29.86, respectively, P < 0.05); the Bcl-2/Bax ratio was lower in group Lep200 as compared to the PI3K specific antagonist with Lep200 group (F = 20.56, P < 0.05). Leptin can significantly inhibit ASMC apoptosis partially via the PI3K/Akt signaling pathway.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

PubMed

Ambrosini, Grazia; Sawle, Ashley D; Musi, Elgilda; Schwartz, Gary K

2015-10-20

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax.

PubMed

Bajpai, R; Matulis, S M; Wei, C; Nooka, A K; Von Hollen, H E; Lonial, S; Boise, L H; Shanmugam, M

2016-07-28

Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM.

Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax

PubMed Central

Bajpai, R; Matulis, SM; Wei, C; Nooka, AK; Von Hollen, HE; Lonial, S; Boise, LH; Shanmugam, M

2016-01-01

Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM. PMID:26640142

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

RACK1 promotes radiation resistance in esophageal cancer via regulating AKT pathway and Bcl-2 expression.

PubMed

Liu, Bowen; Wang, Cong; Chen, Pengxiang; Wang, Lu; Cheng, Yufeng

2017-09-23

RACK1 is a seven Trp-Asp 40 repeat protein, which interacts with a wide range of kinases and proteins. RACK1 plays an important role in the proliferation and progression of various cancers. The aim of this study is to detect the role of RACK1 in the radioresistance in esophageal cancer. The results indicated that downregulation of RACK1 reduced the colony formation ability, proliferation ability and resistance of cells to radiation effection through regulating the radiation-related proteins including pAKT, Bcl-2 and Bim; whereas upregulation of RACK1 promoted the ability and radioresistance of ESCC cells. Our findings suggest that RACK1 promotes proliferation and radioresistance in ESCC cells by activating the AKT pathway, upregulating Bcl-2 expression and downregulating protein levels of Bim. Our study fills in gaps in the field of RACK1 and radiation resistance and may provide new possibilities for improving strategies of radiotherapy in esophageal cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunophenotypic and genetic characteristics of diffuse large B-cell lymphoma in Taiwan.

PubMed

Chang, Sheng-Tsung; Chen, Shang-Wen; Ho, Chung-Han; Kuo, Chun-Chi; Sakata, Seiji; Takeuchi, Kengo; Chuang, Shih-Sung

2016-11-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma type. The immunophenotypic and genetic features of DLBCL in Taiwan have not been characterized. In this study, we performed immunohistochemical analysis and interphase fluorescence in situ hybridization (FISH) using tissue microarray sections to investigate a cohort of unselected DLBCL cases in a single institution in Taiwan from 1990 to 2010. Of the 153 cases investigated, CD10, bcl-6, and MUM1 were expressed in 16.3%, 71.2%, and 71.9% cases, respectively, with 27.5% (n = 42) of cases being classified as having a germinal center B-cell (GCB) origin by the Hans algorithm. By FISH analysis, 19.6%, 4.6%, 26.1%, and 3.9% cases showed rearrangement at IGH, BCL2, BCL6, and MYC loci, respectively, including three (2.0%) cases of double-hit lymphoma. As compared with the non-GCB tumors, GCB tumors more frequently expressed CD10 (p < 0.001) and bcl-6 (p = 0.001) with less frequent expression of MUM1 (p = 0.007). Moreover, GCB tumors more frequently exhibited rearrangement at the BCL2 (p = 0.024) and MYC (p = 0.038) loci than non-GCB tumors. However, there was no survival difference between these two groups. In this first series of DLBCL evaluation from Taiwan, we found that the relative frequency of GCB tumors among DLBCL was low in most East Asian countries. There is a wide range of BCL2 rearrangement rates, higher in the West and lower in East Asia. A larger and/or national study is warranted to better understand the immunophenotypic and molecular features of DLBCL in Taiwan and their respective impact on patient survival. Copyright © 2016. Published by Elsevier B.V.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

PubMed

Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

2016-10-01

Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

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.

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

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

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.

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 action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Activation of PPARβ/δ protects pancreatic β cells from palmitate-induced apoptosis by upregulating the expression of GLP-1 receptor.

PubMed

Yang, Yan; Tong, Yuzhen; Gong, Meng; Lu, Yanrong; Wang, Chengshi; Zhou, Mingliang; Yang, Qiu; Mao, Tingrui; Tong, Nanwei

2014-02-01

We previously showed that activated peroxisome proliferator-activated receptor (PPAR)β/δ can protect pancreatic β cells against lipotoxic apoptosis. However, the molecular mechanism remained unclear. Glucagon-like peptide-1 receptor (GLP-1R) has been reported to exhibit a protective effect against lipotoxic apoptosis in pancreatic β cells. In the present study, we aimed to investigate the underlying molecular mechanisms that PPARβ/δ activation suppressed apoptosis and improved β cell function impaired by fatty acids, focusing on contribution of GLP-1R. Isolated rat islets and rat insulin-secreting INS-1 cells were treated with the PPARβ/δ agonist GW501516 (GW) in the presence or absence of palmitate (PA) and transfected with siRNA for PPARβ/δ or treated with the PPARβ/δ antagonist GSK0660. Apoptosis was assessed by DNA fragmentation, Hoechst 33342 staining and flow cytometry. GLP-1R expression in INS-1 cells and islets was assayed by immunoblotting, quantitative PCR (qPCR) and immunofluorescence staining. SREBP-1c, Caveolin-1, Akt, Bcl-2, Bcl-xl and caspase-3 expression was measured using immunoblotting and qPCR. Our results showed that PPARβ/δ activation decreased apoptosis in β cells and robustly stimulated GLP-1R expression under lipotoxic conditions. GW enhanced glucose-stimulated insulin secretion (GSIS) impaired by PA through stimulation of GLP-1R expression in β cells. Moreover, SREBP-1c/Caveolin-1 signaling was involved in PPARβ/δ-regulated GLP-1R expression. Finally, GW exerted anti-apoptotic effects via interfering with GLP-1R-dependent Akt/Bcl-2 and Bcl-xl/caspase-3 signaling pathways. Our study suggested that the anti-apoptotic action of GW may involve its transcriptional regulation of GLP-1R, and PPARβ/δ activation may represent a new therapeutic method for protecting pancreatic β cells from lipotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Baseline Characteristics Predicting Very Good Outcome of Allogeneic Hematopoietic Cell Transplantation in Young Patients With High Cytogenetic Risk Chronic Lymphocytic Leukemia - A Retrospective Analysis From the Chronic Malignancies Working Party of the EBMT.

PubMed

van Gelder, Michel; Ziagkos, Dimitris; de Wreede, Liesbeth; van Biezen, Anja; Dreger, Peter; Gramatzki, Martin; Stelljes, Matthias; Andersen, Niels Smedegaard; Schaap, Nicolaas; Vitek, Antonin; Beelen, Dietrich; Lindström, Vesa; Finke, Jürgen; Passweg, Jacob; Eder, Matthias; Machaczka, Maciej; Delgado, Julio; Krüger, William; Raida, Luděk; Socié, Gerard; Jindra, Pavel; Afanasyev, Boris; Wagner, Eva; Chalandon, Yves; Henseler, Anja; Schoenland, Stefan; Kröger, Nicolaus; Schetelig, Johannes

2017-10-01

Patients with genetically high-risk relapsed/refractory chronic lymphocytic leukemia have shorter median progression-free survival (PFS) with kinase- and BCL2-inhibitors (KI, BCL2i). Allogeneic hematopoietic stem cell transplantation (alloHCT) may result in sustained PFS, especially in younger patients because of its age-dependent non-relapse mortality (NRM) risk, but outcome data are lacking for this population. Risk factors for 2-year NRM and 8-year PFS were identified in patients < 50 years in an updated European Society for Blood and Marrow Transplantation registry cohort (n = 197; median follow-up, 90.4 months) by Cox regression modeling, and predicted probabilities of NRM and PFS of 2 reference patients with favorable or unfavorable characteristics were plotted. Predictors for poor 8-year PFS were no remission at the time of alloHCT (hazard ratio [HR], 1.7; 95% confidence interval [CI], 1.1-2.5) and partially human leukocyte antigen (HLA)-mismatched unrelated donor (HR, 2.8; 95% CI, 1.5-5.2). The latter variable also predicted a higher risk of 2-year NRM (HR, 4.0; 95% CI, 1.4-11.6) compared with HLA-matched sibling donors. Predicted 2-year NRM and 8-year PFS of a high cytogenetic risk (del(17p) and/or del(11q)) patient in remission with a matched related donor were 12% (95% CI, 3%-22%) and 54% (95% CI, 38%-69%), and for an unresponsive patient with a female partially HLA-matched unrelated donor 37% (95% CI, 12%-62%) and 38% (95% CI, 13%-63%). Low predicted NRM and high 8-year PFS in favorable transplant high cytogenetic risk patients compares favorably with outcomes with KI or BCL2i. Taking into account the amount of uncertainty for predicting survival after alloHCT and after sequential administration of KI and BCL2i, alloHCT remains a valid option for younger patients with high cytogenetic risk chronic lymphocytic leukemia with a well-HLA-matched donor. Copyright © 2017 Elsevier Inc. All rights reserved.

Bcl-2 expression is essential for development and normal physiological properties of tooth hard tissue and saliva production.

PubMed

Saghiri, Mohammad Ali; Asatourian, Armen; Gurel, Zafer; Sorenson, Christine M; Sheibani, Nader

2017-09-15

Apoptosis plays a fundamental role in appropriate tissue development and function. Although expression of Bcl-2 has been reported during tooth and submandibular gland (SMG) development, the physiological role Bcl-2 plays during these processes has not been addressed. This study was performed to evaluate the impact of Bcl-2 expression on the formation and properties of tooth hard tissue, and saliva production. Twenty-four mice (12 males and 12 females) were divided into three groups of eight (n=8): group A (Bcl-2 +/+), group B (Bcl-2 +/-), and group C (Bcl-2 -/-) and subjected to micro-CT analyses. The mineral content of first molars was analyzed by X-Ray diffraction (XRD) and scanning electron microscopy (SEM) color dot map. The surface microhardness was determined by Vickers test on labial surfaces of incisors. Saliva was collected from different groups of mice after subcutaneous injection of pilocarpine. Samples from Bcl-2 -/- mice showed significantly smaller micro-CT values, lower and poor crystallinity of hydroxyapatite (HA), and lowest surface micro hardness. SMG from Bcl-2 -/- mice showed remarkable reduction in size, consistent with reduced saliva accumulation. The absence of Bcl-2 expression in SMG did not affect the expression of other Bcl-2 family members. Thus, Bcl-2 expression influence on the formation and properties of tooth hard tissue, and saliva accumulation. Bcl-2 expression has a significant impact on the mineralogical content of enamel crystals of tooth structure. Lack of Bcl-2 expression led to impaired production of enamel ACP crystals. Copyright © 2017 Elsevier Inc. All rights reserved.

A Model for Long Range Planning for Seminole Community College.

ERIC Educational Resources Information Center

Miner, Norris

A model for long-range planning designed to maximize involvement of college personnel, to improve communication among various areas of the college, to provide a process for evaluation of long-range plans and the planning process, to adjust to changing conditions, to utilize data developed at a level useful for actual operations, and to have…

Biocompatible nanoemulsions based on hemp oil and less surfactants for oral delivery of baicalein with enhanced bioavailability

PubMed Central

Yin, Juntao; Xiang, Cuiyu; Wang, Peiqing; Yin, Yuyun; Hou, Yantao

2017-01-01

Baicalein (BCL) possesses high pharmacological activities but low solubility and stability in the intestinal tract. This study aimed to probe the potential of nanoemulsions (NEs) consisting of hemp oil and less surfactants in ameliorating the oral bioavailability of BCL. BCL-loaded NEs (BCL-NEs) were prepared by high-pressure homogenization technique to reduce the amount of surfactants. BCL-NEs were characterized by particle size, entrapment efficiency (EE), in vitro drug release, and morphology. Bioavailability was studied in Sprague-Dawley rats following oral administration of BCL suspensions, BCL conventional emulsions, and BCL-NEs. The obtained NEs were ~90 nm in particle size with an EE of 99.31%. BCL-NEs significantly enhanced the oral bioavailability of BCL, up to 524.7% and 242.1% relative to the suspensions and conventional emulsions, respectively. BCL-NEs exhibited excellent intestinal permeability and transcellular transport ability. The cytotoxicity of BCL-NEs was documented to be low and acceptable for oral purpose. Our findings suggest that such novel NEs and preparative process provide a promising alternative to current formulation technologies and suitable for oral delivery of drugs with bioavailability issues. PMID:28435268

Biocompatible nanoemulsions based on hemp oil and less surfactants for oral delivery of baicalein with enhanced bioavailability.

PubMed

Yin, Juntao; Xiang, Cuiyu; Wang, Peiqing; Yin, Yuyun; Hou, Yantao

2017-01-01

Baicalein (BCL) possesses high pharmacological activities but low solubility and stability in the intestinal tract. This study aimed to probe the potential of nanoemulsions (NEs) consisting of hemp oil and less surfactants in ameliorating the oral bioavailability of BCL. BCL-loaded NEs (BCL-NEs) were prepared by high-pressure homogenization technique to reduce the amount of surfactants. BCL-NEs were characterized by particle size, entrapment efficiency (EE), in vitro drug release, and morphology. Bioavailability was studied in Sprague-Dawley rats following oral administration of BCL suspensions, BCL conventional emulsions, and BCL-NEs. The obtained NEs were ~90 nm in particle size with an EE of 99.31%. BCL-NEs significantly enhanced the oral bioavailability of BCL, up to 524.7% and 242.1% relative to the suspensions and conventional emulsions, respectively. BCL-NEs exhibited excellent intestinal permeability and transcellular transport ability. The cytotoxicity of BCL-NEs was documented to be low and acceptable for oral purpose. Our findings suggest that such novel NEs and preparative process provide a promising alternative to current formulation technologies and suitable for oral delivery of drugs with bioavailability issues.

Butyric acid induces apoptosis via oxidative stress in Jurkat T-cells.

PubMed

Kurita-Ochiai, T; Ochiai, K

2010-07-01

Reactive oxygen species (ROS) are essential for the induction of T-cell apoptosis by butyric acid, an extracellular metabolite of periodontopathic bacteria. To determine the involvement of oxidative stress in apoptosis pathways, we investigated the contribution of ROS in mitochondrial signaling pathways, death-receptor-initiated signaling pathway, and endoplasmic reticulum stress in butyric-acid-induced T-cell apoptosis. N-acetyl-L-Cysteine (NAC) abrogated mitochondrial injury, cytochrome c, AIF, and Smac release, and Bcl-2 and Bcl-xL suppression and Bax and Bad activation induced by butyric acid. However, the decrease in cFLIP expression by butyric acid was not restored by treatment with NAC; increases in caspase-4 and -10 activities by butyric acid were completely abrogated by NAC. NAC also affected the elevation of GRP78 and CHOP/GADD153 expression by butyric acid. These results suggest that butyric acid is involved in mitochondrial-dysfunction- and endoplasmic reticulum stress-mediated apoptosis in human Jurkat T-cells via a ROS-dependent mechanism.

Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2.

PubMed

Ahmadian, Elham; Khosroushahi, Ahmad Yari; Eftekhari, Aziz; Farajnia, Safar; Babaei, Hossein; Eghbal, Mohammad Ali

2018-03-01

Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72?h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay11- 7082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box

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

Liang Xin; Xu Ke; Xu Yufang

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report heremore » that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P{sub 2} promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research Highlights: > B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. > B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. > B1 induced significant increase of p53 binding to Bcl-2 P{sub 2} promoter TATA box.« less

ANXA11 regulates the tumorigenesis, lymph node metastasis and 5-fluorouracil sensitivity of murine hepatocarcinoma Hca-P cells by targeting c-Jun.

PubMed

Liu, Shuqing; Guo, Chunmei; Wang, Jiasheng; Wang, Bo; Qi, Houbao; Sun, Ming-Zhong

2016-03-29

Annexin A11 (Anxa11) is associated with various cancers. Using a pair of syngeneic murine hepatocarcinoma cells, Hca-P with ~25% and Hca-F with ~75% lymph node metastatic (LNM) potentials, we demonstrated Anxa11 involvement in hepatocarcinoma lymphatic metastasis. Here, ANXA11 acted as a suppressor for the tumorigenicity, LNM and 5-FU resistance of Hca-P via c-Jun. We constructed monoclonal Hca-P cell line with stable ANXA11 knockdown. Although Bax and Bcl-2 levels increased in shRNA-Anxa11-transfected Hca-P, ANXA11 downregulation showed no clear effect on Hca-P apoptosis. ANXA11 downregulation promoted in vitro migration and invasion capacities of Hca-P. In situ adhesion potential of Hca-P cells toward LN was significantly enhanced following ANXA11 downregulation. Consistently, ANXA11 downregulation promoted the in vivo tumor growth and LNM capacities of Hca-P cells. ANXA11 knockdown enhanced the chemoresistance of Hca-P cells specifically toward 5-FU instead of cisplatin. Its downregulation increased c-Jun (pSer73) and decreased c-Jun (pSer243) levels in Hca-P. c-Jun (pSer243) downregulation seemed to be only correlated with ANXA11 knockdown without the connection to 5-FU treatment. Interestingly, compared with scramble-Hca-P cells, the levels of c-Jun and c-Jun (pSer73) in shRNA-Anxa11-Hca-P cells were upregulated in the presences of 0.1 and 1.0 mg/L 5-FU. The levels changes from c-Jun and c-Jun (pSer73) in Hca-P cells showed a more obvious tendency with the combination of ANXA11 knockdown and 5-FU treatment. ANXA11 level regulates LNM and 5-FU resistance of Hca-P via c-Jun pathway. It might play an important role in hepatocarcinoma cell malignancy and be a therapeutic target for hepatocarcinoma.

ANXA11 regulates the tumorigenesis, lymph node metastasis and 5-fluorouracil sensitivity of murine hepatocarcinoma Hca-P cells by targeting c-Jun

PubMed Central

Wang, Bo; Qi, Houbao; Sun, Ming-Zhong

2016-01-01

Annexin A11 (Anxa11) is associated with various cancers. Using a pair of syngeneic murine hepatocarcinoma cells, Hca-P with ~25% and Hca-F with ~75% lymph node metastatic (LNM) potentials, we demonstrated Anxa11 involvement in hepatocarcinoma lymphatic metastasis. Here, ANXA11 acted as a suppressor for the tumorigenicity, LNM and 5-FU resistance of Hca-P via c-Jun. We constructed monoclonal Hca-P cell line with stable ANXA11 knockdown. Although Bax and Bcl-2 levels increased in shRNA-Anxa11-transfected Hca-P, ANXA11 downregulation showed no clear effect on Hca-P apoptosis. ANXA11 downregulation promoted in vitro migration and invasion capacities of Hca-P. In situ adhesion potential of Hca-P cells toward LN was significantly enhanced following ANXA11 downregulation. Consistently, ANXA11 downregulation promoted the in vivo tumor growth and LNM capacities of Hca-P cells. ANXA11 knockdown enhanced the chemoresistance of Hca-P cells specifically toward 5-FU instead of cisplatin. Its downregulation increased c-Jun (pSer73) and decreased c-Jun (pSer243) levels in Hca-P. c-Jun (pSer243) downregulation seemed to be only correlated with ANXA11 knockdown without the connection to 5-FU treatment. Interestingly, compared with scramble-Hca-P cells, the levels of c-Jun and c-Jun (pSer73) in shRNA-Anxa11-Hca-P cells were upregulated in the presences of 0.1 and 1.0 mg/L 5-FU. The levels changes from c-Jun and c-Jun (pSer73) in Hca-P cells showed a more obvious tendency with the combination of ANXA11 knockdown and 5-FU treatment. ANXA11 level regulates LNM and 5-FU resistance of Hca-P via c-Jun pathway. It might play an important role in hepatocarcinoma cell malignancy and be a therapeutic target for hepatocarcinoma. PMID:26908448

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 the inhibition of proliferation and cancer cell resistance to apoptosis.« less

Dual targeting of MDM2 and BCL2 as a therapeutic strategy in neuroblastoma.

PubMed

Van Goethem, Alan; Yigit, Nurten; Moreno-Smith, Myrthala; Vasudevan, Sanjeev A; Barbieri, Eveline; Speleman, Frank; Shohet, Jason; Vandesompele, Jo; Van Maerken, Tom

2017-08-22

Wild-type p53 tumor suppressor activity in neuroblastoma tumors is hampered by increased MDM2 activity, making selective MDM2 antagonists an attractive therapeutic strategy for this childhood malignancy. Since monotherapy in cancer is generally not providing long-lasting clinical responses, we here aimed to identify small molecule drugs that synergize with idasanutlin (RG7388). To this purpose we evaluated 15 targeted drugs in combination with idasanutlin in three p53 wild type neuroblastoma cell lines and identified the BCL2 inhibitor venetoclax (ABT-199) as a promising interaction partner. The venetoclax/idasanutlin combination was consistently found to be highly synergistic in a diverse panel of neuroblastoma cell lines, including cells with high MCL1 expression levels. A more pronounced induction of apoptosis was found to underlie the synergistic interaction, as evidenced by caspase-3/7 and cleaved PARP measurements. Mice carrying orthotopic xenografts of neuroblastoma cells treated with both idasanutlin and venetoclax had drastically lower tumor weights than mice treated with either treatment alone. In conclusion, these data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy in neuroblastoma.

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.

Genome-Wide Association Study of the Modified Stumvoll Insulin Sensitivity Index Identifies BCL2 and FAM19A2 as Novel Insulin Sensitivity Loci

PubMed Central

Gustafsson, Stefan; Rybin, Denis; Stančáková, Alena; Chen, Han; Liu, Ching-Ti; Hong, Jaeyoung; Jensen, Richard A.; Rice, Ken; Morris, Andrew P.; Mägi, Reedik; Tönjes, Anke; Prokopenko, Inga; Kleber, Marcus E.; Delgado, Graciela; Silbernagel, Günther; Jackson, Anne U.; Appel, Emil V.; Grarup, Niels; Lewis, Joshua P.; Montasser, May E.; Landenvall, Claes; Staiger, Harald; Luan, Jian’an; Frayling, Timothy M.; Weedon, Michael N.; Xie, Weijia; Morcillo, Sonsoles; Martínez-Larrad, María Teresa; Biggs, Mary L.; Chen, Yii-Der Ida; Corbaton-Anchuelo, Arturo; Færch, Kristine; Gómez-Zumaquero, Juan Miguel; Goodarzi, Mark O.; Kizer, Jorge R.; Koistinen, Heikki A.; Leong, Aaron; Lind, Lars; Lindgren, Cecilia; Machicao, Fausto; Manning, Alisa K.; Martín-Núñez, Gracia María; Rojo-Martínez, Gemma; Rotter, Jerome I.; Siscovick, David S.; Zmuda, Joseph M.; Zhang, Zhongyang; Serrano-Rios, Manuel; Smith, Ulf; Soriguer, Federico; Hansen, Torben; Jørgensen, Torben J.; Linnenberg, Allan; Pedersen, Oluf; Walker, Mark; Langenberg, Claudia; Scott, Robert A.; Wareham, Nicholas J.; Fritsche, Andreas; Häring, Hans-Ulrich; Stefan, Norbert; Groop, Leif; O’Connell, Jeff R.; Boehnke, Michael; Bergman, Richard N.; Collins, Francis S.; Mohlke, Karen L.; Tuomilehto, Jaakko; März, Winfried; Kovacs, Peter; Stumvoll, Michael; Psaty, Bruce M.; Kuusisto, Johanna; Laakso, Markku; Meigs, James B.; Dupuis, Josée; Ingelsson, Erik; Florez, Jose C.

2016-01-01

Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: rs13422522 (NYAP2; P = 8.87 × 10−11), rs12454712 (BCL2; P = 2.7 × 10−8), and rs10506418 (FAM19A2; P = 1.9 × 10−8). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci. PMID:27416945

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth

PubMed Central

Renton, John P.; Xu, Ningyong; Clark, J. Jason; Hansen, Marlan R.

2012-01-01

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl-2 prevents SGN death, but inhibits neurite growth. Here we assessed the consequences of Bcl-2 targeted to either the mitochondria (GFP-Bcl-2-Maob) or endoplasmic reticulum (ER, GFP-Bcl-2-Cb5) on cultured SGN survival and neurite growth. Transfection of wild type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob increased SGN survival, with GFP-Bcl-2-Cb5 providing the most robust response. Paradoxically, expression of GFP-Bcl-2-Maob results in SGN death in the presence of neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears specific for neurotrophin signaling, since co-expression of constitutively active mitogen activated kinase kinase (MEKΔEE) or phosphatidyl inositol-3 kinase (P110), but not other prosurvival stimuli (e.g. membrane depolarization), also results in the loss of SGNs expressing GFP-Bcl-2-Maob. MEKΔEE and P110 promote SGN survival while P110 promotes neurite growth to a greater extent than NT-3 or MEKΔEE. However wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5 and GFP-Bcl-2-Maob inhibit neurite growth even in the presence of neurotrophins, MEKΔEE, or P110. Historically, Bcl-2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl-2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl-2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. PMID:20209634

Hsp27 (HSPB1) differential expression in normal salivary glands and pleomorphic adenomas and association with an increased Bcl2/Bax ratio.

PubMed

Siqueira, Elisa C de; Souza, Fabrício T A; Diniz, Marina G; Gomez, Ricardo S; Gomes, Carolina C

2015-01-01

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. The Hsp27 (HSPB1) is an antiapoptotic protein whose synthesis follows cytotoxic stresses and result in a transient increase in tolerance to subsequent cell injury. Although Hsp27 is expressed in a range of normal tissues and neoplasms, a wide variation in its expression exists among different cells and tissues types. In certain tumours of glandular origin (such as oesophageal adenocarcinomas), the level of Hsp27 is decreased. In the present study, Hsp27 protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) in a set of 18 fresh PA and 12 normal salivary gland samples. In addition, we tested if Hsp27 protein levels correlated with p53 expression and cell proliferation index, as well as with the transcriptional levels of Bcl-2-associated X protein (BAX), B cell lymphoma 2 (BCL2) and Caspase 3 in PA. We further tested the association between Hsp27 expression and PA tumour size. While all normal salivary gland samples expressed Hsp27 protein, only half of the PA samples expressed it, resulting in a reduced expression of Hsp27 in PA when compared with normal salivary glands (P = 0.003). The expression levels of this protein correlated positively with a higher messenger ribonucleic acid (mRNA) ratio of Bcl2/Bax (R = 0.631; P = 0.01). In conclusion, a decreased Hsp27 protein expression level in PA was found. In addition, Hsp27 levels correlated positively with the Bcl2/Bax mRNA ratio, suggesting an antiapoptotic effect.

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/.

Assessment of global ischemic/reperfusion and Tacrolimus administration on CA1 region of hippocampus: gene expression profiles of BAX and BCL2 genes.

PubMed

Badr, R; Hashemi, M; Javadi, G; Movafagh, A; Mahdian, R

2016-01-01

It is well known that hippocampus has a pivotal role in learning, formation and consolidation of memory. Global cerebral ischemia causes severe damage to pyramidal neurons of the CA1 region and usually results in residual neurological deficits following a recovery from ischemia. Scientists investigate to find the molecular mechanism of apoptosis and to use this cell death for clinical treatment. In this investigation, we evaluated the molecular mechanism of FK-506 in apoptosis using gene expression quantification of BAX and BCL-2 genes in hippocampus following global ischemic/reperfusion. In the present experimental study, adult male Wistar rats were obtained and housed under standard conditions. Each experimental group consisted of five rats and was equally distributed in the normal control, ischemia/reperfusion, ischemia/reperfusion followed by FK-506. Global ischemia was induced for animals in ischemia and drug groups. In the drug group, moreover, two doses of FK-506 were injected as IV injection and intra-peritoneal (IP) injection after 48 h. Then, hippocampus tissue was removed. Consequently, RNA isolated, cDNA was synthesized and Real-Time PCR was performed. Finally, the obtained data was analyzed statistically (p<0.05). The quantitative results showed the BAX expression ratio increased approximately 3-times in ischemia/reperfusion (3.11 ± 0.28) group compared to the untreated (NR) and the drug group (p<0.001). The statistical analysis showed a significant difference for BCL-2 expression between the experimental groups (p<0.001). The mRNA level of BCL-2 decreased in the ischemia/reperfusion group, while it was without alteration in the other groups. The results showed that global cerebral ischemia/reperfusion induced BAX as pro-apoptotic gene and tacrolimus a neuroprotective drug inhibited BAX gene expression and induced BCL-2 gene expression as anti-apoptotic gene (Tab. 2, Fig. 3, Ref. 21).

Sulforaphane-induced apoptosis in Xuanwei lung adenocarcinoma cell line XWLC-05.

PubMed

Zhou, Lan; Yao, Qian; Li, Yan; Huang, Yun-Chao; Jiang, Hua; Wang, Chuan-Qiong; Fan, Lei

2017-01-01

Xuanwei district in Yunnan Province has the highest incidence of lung cancer in China, especially among non-smoking women. Cruciferous vegetables can reduce lung cancer risk by prompting a protective mechanism against respiratory tract inflammation caused by air pollution, and are rich in sulforaphane, which can induce changes in gene expression. We investigated the effect of sulforaphane-induced apoptosis in Xuanwei lung adenocarcinoma cell line (XWCL-05) to explore the value of sulforaphane in lung cancer prevention and treatment. Cell growth inhibition was determined by methyl thiazolyl tetrazolium assay; cell morphology and apoptosis were observed under transmission electron microscope; cell cycle and apoptosis rates were detected using flow cytometry; B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax) messenger RNA expression were determined by quantitative PCR; and p53, p73, p53 upregulated modulator of apoptosis (PUMA), Bax, Bcl-2, and caspase-9 protein expression were detected by Western blotting. Sulforaphane inhibited XWLC-05 cell growth with inhibitory concentration (IC) 50 of 4.04, 3.38, and 3.02 μg/mL at 24, 48, and 72 hours, respectively. Sulforaphane affected the XWLC-05 cell cycle as cells accumulated in the G2/M phase. The proportion of apoptotic cells observed was 27.6%. Compared with the control, the sulforaphane group showed decreased Bcl-2 and p53 expression, and significantly increased p73, PUMA, Bax, and caspase-9 protein expression (P < 0.05). Sulforaphane induces Xuanwei lung adenocarcinoma cell apoptosis. Its possible mechanism may involve the upregulation of p73 expression and its effector target genes PUMA and Bax in lung cancer cells, downregulation of the anti-apoptotic gene B cl -2, and activation of caspase-9. It may also involve downregulation of the mutant p53 protein. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Shoulder and Elbow Recovery at 2 and 11 Years Following Brachial Plexus Reconstruction.

PubMed

Wang, Jung-Pan; Rancy, Schneider K; Lee, Steve K; Feinberg, Joseph H; Wolfe, Scott W

2016-02-01

To report short-term and long-term outcomes on a single patient cohort observed longitudinally after nerve reconstruction for adult brachial plexus injury. Eleven male patients who underwent plexus reconstruction by the same surgeon at 2 institutions presented for clinical examination 7.5 or more years after surgery (average, 11.4 years; range, 7.5-22 years). Average age at the time of operation was 35 years (range, 17-73 years). Mean delay until surgery was 5 months (range, 2-11 months). Two patients had C5 paralysis, 2 had C5-C6 paralysis, 2 had C5-C7 paralysis, and 5 had complete 5-level injuries. Outcome parameters included active range of motion (ROM) in degrees, a modified British Medical Research Council (mBMRC) scale for muscle strength, and electromyographic motor unit configuration and recruitment pattern. Differences in ROM and mBMRC between 2-year and long-term follow-up were assessed with paired-sample t tests using an alpha value of .05. Average shoulder abduction and mBMRC at final follow-up were both significantly improved compared with the 2-year follow-up results (P < .05). Average elbow flexion and mBMRC increased significantly between 2 years and final follow-up (P < .05). Electromyographic results for 6 patients at final follow-up showed improved motor unit configuration in 10 of 15 muscles and improved recruitment in 3 of 15 muscles compared with 2-year electromyographic results. Patients continued to gain ROM and strength in the shoulder and elbow well after 2 to 3 years after surgery, contrary to previous reports. Although the precise mechanism is unknown, we speculate that a number of factors may be involved, including terminal collateral sprouting, maturation of motor units, improvements in motor unit recruitment, additional muscle fiber hypertrophy, or an as-yet undescribed mechanism. We recommend that patients be encouraged to continue strengthening exercises well after the initial recovery period and that more comparative long-term data be collected to expand on these observations. Copyright © 2016 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

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.

Triple Hit Lymphoma: Rare Cases With Less Dire Than Usual Prognosis.

PubMed

Kallen, Michael E; Alexanian, Serge; Said, Jonathan; Quintero-Rivera, Fabiola

2016-12-01

Triple hit lymphomas are a subset of so-called double hit non-Hodgkin lymphomas exhibiting simultaneous gene translocations/disruption of MYC, BCL2, and BCL6; however, their overlapping morphologic features and complex genetic rearrangements can render classification and prognostication vexing. Clinically triple hit lymphomas are thought to demonstrate aggressive behavior, similar to or worse than that of double hit lymphomas. Only rare reports of long term survivors exist and raise the possibility that unidentified morphologic, immunologic, or cytogenetic differences may impart a less adverse prognosis than current literature and opinion may suggest. Here we report 3 such cases with less aggressive behavior. Cases such as these may prove useful in comparing outcomes, and underlying mechanisms of tumor progression, in aggressive non-Hodgkin lymphomas. © The Author(s) 2016.

Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

PubMed

Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

2016-01-01

Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

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.

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

Empirical mode decomposition and long-range correlation analysis of sunspot time series

NASA Astrophysics Data System (ADS)

Zhou, Yu; Leung, Yee

2010-12-01

Sunspots, which are the best known and most variable features of the solar surface, affect our planet in many ways. The number of sunspots during a period of time is highly variable and arouses strong research interest. When multifractal detrended fluctuation analysis (MF-DFA) is employed to study the fractal properties and long-range correlation of the sunspot series, some spurious crossover points might appear because of the periodic and quasi-periodic trends in the series. However many cycles of solar activities can be reflected by the sunspot time series. The 11-year cycle is perhaps the most famous cycle of the sunspot activity. These cycles pose problems for the investigation of the scaling behavior of sunspot time series. Using different methods to handle the 11-year cycle generally creates totally different results. Using MF-DFA, Movahed and co-workers employed Fourier truncation to deal with the 11-year cycle and found that the series is long-range anti-correlated with a Hurst exponent, H, of about 0.12. However, Hu and co-workers proposed an adaptive detrending method for the MF-DFA and discovered long-range correlation characterized by H≈0.74. In an attempt to get to the bottom of the problem in the present paper, empirical mode decomposition (EMD), a data-driven adaptive method, is applied to first extract the components with different dominant frequencies. MF-DFA is then employed to study the long-range correlation of the sunspot time series under the influence of these components. On removing the effects of these periods, the natural long-range correlation of the sunspot time series can be revealed. With the removal of the 11-year cycle, a crossover point located at around 60 months is discovered to be a reasonable point separating two different time scale ranges, H≈0.72 and H≈1.49. And on removing all cycles longer than 11 years, we have H≈0.69 and H≈0.28. The three cycle-removing methods—Fourier truncation, adaptive detrending and the proposed EMD-based method—are further compared, and possible reasons for the different results are given. Two numerical experiments are designed for quantitatively evaluating the performances of these three methods in removing periodic trends with inexact/exact cycles and in detecting the possible crossover points.

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas.

PubMed

O'Steen, Shyril; Green, Damian J; Gopal, Ajay K; Orozco, Johnnie J; Kenoyer, Aimee L; Lin, Yukang; Wilbur, D Scott; Hamlin, Donald K; Fisher, Darrell R; Hylarides, Mark D; Gooley, Theodore A; Waltman, Amelia; Till, Brian G; Press, Oliver W

2017-07-15

Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR . ©2017 American Association for Cancer Research.

Promising efficacy and acceptable safety of venetoclax plus bortezomib and dexamethasone in relapsed/refractory MM.

PubMed

Moreau, Philippe; Chanan-Khan, Asher; Roberts, Andrew W; Agarwal, Amit B; Facon, Thierry; Kumar, Shaji; Touzeau, Cyrille; Punnoose, Elizabeth A; Cordero, Jaclyn; Munasinghe, Wijith; Jia, Jia; Salem, Ahmed Hamed; Freise, Kevin J; Leverson, Joel D; Enschede, Sari Heitner; Ross, Jeremy A; Maciag, Paulo C; Verdugo, Maria; Harrison, Simon J

2017-11-30

The antiapoptotic proteins BCL-2 and myeloid cell leukemia sequence 1 (MCL-1) promote multiple myeloma (MM) cell survival. Venetoclax is a selective, orally bioavailable small-molecule BCL-2 inhibitor; bortezomib can indirectly inhibit MCL-1. In preclinical studies, venetoclax enhanced bortezomib activity, suggesting that cotargeting of BCL-2 and MCL-1 could be an effective treatment strategy in myeloma. This phase 1b trial studied patients with relapsed/refractory MM receiving daily venetoclax (50-1200 mg per designated dose cohort; 800 mg in safety expansion) in combination with bortezomib and dexamethasone. A total of 66 patients were enrolled (54 in the dose-escalation cohorts and 12 in the safety expansion). Patients had received a median of 3 prior therapies (range, 1-13); 26 (39%) were refractory to prior bortezomib and 35 (53%) to lenalidomide; 39 (59%) had prior stem cell transplant. The combination was generally well tolerated, and common adverse events included mild gastrointestinal toxicities (diarrhea [46%], constipation [41%], and nausea [38%]) and grade 3/4 cytopenias (thrombocytopenia [29%] and anemia [15%]). The overall response rate (ORR) was 67% (44/66); 42% achieved very good partial response or better (≥VGPR). Median time to progression and duration of response were 9.5 and 9.7 months, respectively. ORR of 97% and ≥VGPR 73% were seen in patients not refractory to bortezomib who had 1 to 3 prior therapies. Patients with high BCL2 expression had a higher ORR (94% [17/18]) than patients with low BCL2 expression (59% [16/27]). This novel combination of venetoclax with bortezomib and dexamethasone has an acceptable safety profile and promising efficacy in patients with relapsed/refractory MM. This trial was registered at www.clinicaltrials.gov as #NCT01794507. © 2017 by The American Society of Hematology.

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.

The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells.

PubMed

Lian, Jiqin; Karnak, David; Xu, Liang

2010-11-01

Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737.

PubMed

Mani, Jens; Antonietti, Patrick; Rakel, Stefanie; Blaheta, Roman; Bartsch, Georg; Haferkamp, Axel; Kögel, Donat

2016-02-01

BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers. Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (-)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting. Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737. Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

Effects of Renal Ischemic Postconditioning on Myocardial Ultrastructural Organization and Myocardial Expression of Bcl-2/Bax in Rabbits

PubMed Central

Zhang, Wen-zhong; Li, Rong; Liu, Song; Ning, Xian-feng; Cai, Shang-lang

2016-01-01

We investigated the cardioprotective effect of renal ischemic postconditioning (RI-PostC) and its mechanisms in a rabbit model. Rabbits underwent 60 min of left anterior descending coronary artery occlusion (LADO) and 6 h of reperfusion. The ischemia-reperfusion (IR) group underwent LADO and reperfusion only. In the RI-PostC group, the left renal artery underwent 3 cycles of occlusion for 30 seconds and release for 30 seconds, before the coronary artery was reperfused. In the RI-PostC + GF109203X group, the rabbits received 0.05 mg/kg GF109203X (protein kinase C inhibitor) intravenously for 10 min followed by RI-PostC. Light microscopy and electron microscopy demonstrated that the RI-PostC group showed less pronounced changes, a smaller infarct region, and less apoptosis than the other two groups. Bcl-2 and Bax protein expression did not differ between the IR and RI-PostC + GF109203X groups. However, in the RI-PostC group, Bcl-2 protein expression was significantly higher and Bax protein expression was significantly lower than in the other two groups (P < 0.05). Changes in heart rate and mean arterial pressure were also smaller in the RI-PostC group than in the other two groups. These results indicate that RI-PostC can ameliorate myocardial ischemia-reperfusion injury and increase the Bcl-2/Bax ratio through a mechanism involving protein kinase C. PMID:28097153

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.

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.

Complex inhibition of autophagy by mitochondrial aldehyde dehydrogenase shortens lifespan and exacerbates cardiac aging.

PubMed

Zhang, Yingmei; Wang, Cong; Zhou, Jingmin; Sun, Aijun; Hueckstaedt, Lindsay K; Ge, Junbo; Ren, Jun

2017-08-01

Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a cascade of biological processes including aging. A number of autophagy regulators have been identified. Here we demonstrated that mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme with the most common single point mutation in humans, governs cardiac aging through regulation of autophagy. Myocardial mechanical and autophagy properties were examined in young (4months) and old (26-28months) wild-type (WT) and global ALDH2 transgenic mice. ALDH2 overexpression shortened lifespan by 7.7% without affecting aging-associated changes in plasma metabolic profiles. Myocardial function was compromised with aging associated with cardiac hypertrophy, the effects were accentuated by ALDH2. Aging overtly suppressed autophagy and compromised autophagy flux, the effects were exacerbated by ALDH2. Aging dampened phosphorylation of JNK, Bcl-2, IKKβ, AMPK and TSC2 while promoting phosphorylation of mTOR, the effects of which were exaggerated by ALDH2. Co-immunoprecipitation revealed increased dissociation between Bcl-2 and Beclin-1 (result of decreased Bcl-2 phosphorylation) in aging, the effect of which was exacerbated with ALDH2. Chronic treatment of the autophagy inducer rapamycin alleviated aging-induced cardiac dysfunction in both WT and ALDH2 mice. Moreover, activation of JNK and inhibition of either Bcl-2 or IKKβ overtly attenuated ALDH2 activation-induced accentuation of cardiomyocyte aging. Examination of the otherwise elderly individuals revealed a positive correlation between cardiac function/geometry and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may suppress myocardial autophagy possibly through a complex JNK-Bcl-2 and IKKβ-AMPK-dependent mechanism en route to accentuation of myocardial remodeling and contractile dysfunction in aging. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang. Copyright © 2017 Elsevier B.V. All rights reserved.

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

GESTATIONAL DIABETES MELLITUS ALTERS APOPTOTIC AND INFLAMMATORY GENE EXPRESSION OF TROPHOBASTS FROM HUMAN TERM PLACENTA

PubMed Central

MAGEE, Thomas R.; ROSS, Michael G.; WEDEKIND, Lauren; DESAI, Mina; KJOS, Siri; BELKACEMI, Louiza

2014-01-01

AIM Increased placental growth secondary to reduced apoptosis may contribute to the development of macrosomia in GDM pregnancies. We hypothesize that reduced apoptosis in GDM placentas is caused by dysregulation of apoptosis related genes from death receptors or mitochondrial pathway or both to enhance placental growth in GDM pregnancies. METHODS Newborn and placental weights from women with no pregnancy complications (controls; N=5), or with GDM (N=5) were recorded. Placental villi from both groups were either fixed for TUNEL assay, or snap frozen for gene expression analysis by apoptosis PCR microarrays and qPCR. RESULTS Maternal, placental and newborn weights were significantly higher in the GDM group vs. Controls. Apoptotic index of placentas from the GDM group was markedly lower than the Controls. At a significant threshold of 1.5, seven genes (BCL10, BIRC6, BIRC7, CASP5, CASP8P2, CFLAR, and FAS) were down regulated, and 13 genes (BCL2, BCL2L1, BCL2L11, CASP4, DAPK1, IκBκE, MCL1, NFκBIZ, NOD1, PEA15, TNF, TNFRSF25, and XIAP) were unregulated in the GDM placentas. qPCR confirmed the consistency of the PCR microarray. Using Western blotting we found significantly decreased placental pro-apoptotic FAS receptor and FAS ligand (FASL), and increased mitochondrial anti-apoptotic BCL2 post GDM insult. Notably, caspase-3, which plays a central role in the execution-phase of apoptosis, and its substrate poly (ADP-ribose) polymerase (PARP) were significantly down regulated in GDM placentas, as compared to non-diabetic Control placentas. CONCLUSION . Women with gestational diabetes (GDM) are at increased risk for having macrosomic newborns, and larger placentas with reduced apoptosis. Decreased apoptosis subsequent to alterations in apoptotic and inflammatory genes may promote elevated weight in the GDM placentas. PMID:24768206

Increased lymphocyte apoptosis in mouse models of colitis upon ABT-737 treatment is dependent upon BIM expression.

PubMed

Lutz, C; Mozaffari, M; Tosevski, V; Caj, M; Cippà, P; McRae, B L; Graff, C L; Rogler, G; Fried, M; Hausmann, M

2015-08-01

Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD. © 2015 British Society for Immunology.

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.

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.

Flow cytometry in the bone marrow evaluation of follicular and diffuse large B-cell lymphomas.

PubMed

Palacio, C; Acebedo, G; Navarrete, M; Ruiz-Marcellán, C; Sanchez, C; Blanco, A; López, A

2001-09-01

Bone marrow biopsies are routinely performed in the staging of patients with lymphoma. Despite the lack of evidence for its usefulness, many institutions include flow cytometry (FC) of bone-marrow aspirates in an attempt to increase sensitivity and specificity. The aim of this study is to evaluate the usefulness of FC for the assessment of bone-marrow involvement by lymphoma in follicular (FL) and diffuse large B-cell lymphomas (DLBCL). Seventy-nine bone marrow biopsies from 65 patients diagnosed with FL or DLBCL were examined to compare histology and FC for the assessment of bone-marrow involvement by lymphoma. Bone marrow histology showed involvement (BM+) in 16 cases (20.3%), lack of infiltration (BM(-)) in 52 cases (65.8%) and undetermined or undiagnosed for involvement (BMu) in 11 cases (13.9%). FC was positive for involvement in 28 cases (35.4%) and negative in 51 cases (64.6%). 65 cases (95%) showed concordance between the results of morphology and FC (BM(+)/FC(+) or BM(-)/FC(-)). No BM(+)/FC(-) cases were observed. 3 cases showed discrepant results (BM(-)/FC(+)). In these 3 cases the molecular studies (PCR) demonstrated clonal rearrangement of the heavy immunoglobulin chain (IgH) and/or bcl2-IgH in agreement with the flow results. Among the 11 cases with BMu, all but 2 were FC(+) and concordance with the PCR results was seen in 9 cases (81.9%). We conclude that FC is just as sensitive or perhaps slightly more sensitive than histology in the detection of bone marrow involvement in FL and DLBCL. FC studies may be warranted in those cases in which the morphology is not diagnosed. The clinical relevance of the small clonal B-cell population in patients without histologic bone marrow involvement (BM(-)/FC(+) cases) remains an open question.

[Apoptosis of human lung carcinoma cell line GLC-82 induced by high power electromagnetic pulse].

PubMed

Cao, Xiao-zhe; Zhao, Mei-lan; Wang, De-wen; Dong, Bo

2002-09-01

Electromagnetic pulse (EMP) could be used for sterilization of food and the efficiency is higher than 2450 MHz continuous microwave done. This study was designed to evaluate the effect of electromagnetic pulse (EMP) on apoptosis of human lung carcinoma cell line GLC-82, so that to explore and develop therapeutic means for cancer. The injury changes in GLC-82 cells after irradiated with EMP (electric field intensity was 60 kV/m, 5 pulses/2 min) were analyzed by cytometry, MTT chronometry, and flow cytometry. The immunohistochemical SP staining was used to determine the expressions of bcl-2 protein and p53 protein. The stained positive cells were analyzed by CMIAS-II image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. EMP could obviously inhibited proliferation and activity of lung carcinoma cell line GLC-82. The absorbance value (A570) of MTT decreased immediately, at 0 h, 1 h, and 6 h after the GLC-82 cells irradiated by EMP as compared with control group. The highest apoptosis rate was found to reach 13.38% by flow cytometry at 6 h after EMP irradiation. Down-regulation of bcl-2 expression and up-regulation of p53 expression were induced by EMP. EMP promotes apoptosis of GLC-82 cells. At same time, EMP can down-regulate bcl-2 expression and up-regulate p53 expression in GLC-82 cells. The bcl-2 and the p53 protein may involve the apoptotic process.

The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

PubMed

Clipson, Alexandra; Barrans, Sharon; Zeng, Naiyan; Crouch, Simon; Grigoropoulos, Nicholas F; Liu, Hongxiang; Kocialkowski, Sylvia; Wang, Ming; Huang, Yuanxue; Worrillow, Lisa; Goodlad, John; Buxton, Jenny; Neat, Michael; Fields, Paul; Wilkins, Bridget; Grant, John W; Wright, Penny; Ei-Daly, Hesham; Follows, George A; Roman, Eve; Watkins, A James; Johnson, Peter W M; Jack, Andrew; Du, Ming-Qing

2015-07-01

A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP ( n  = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP ( n  = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

PubMed Central

Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Zou, Lihua; Gould, Joshua; Saksena, Gordon; Stransky, Nicolas; Rangel-Escareño, Claudia; Fernandez-Lopez, Juan Carlos; Hidalgo-Miranda, Alfredo; Melendez-Zajgla, Jorge; Hernández-Lemus, Enrique; Schwarz-Cruz y Celis, Angela; Imaz-Rosshandler, Ivan; Ojesina, Akinyemi I.; Jung, Joonil; Pedamallu, Chandra S.; Lander, Eric S.; Habermann, Thomas M.; Cerhan, James R.; Shipp, Margaret A.; Getz, Gad; Golub, Todd R.

2012-01-01

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease. PMID:22343534

Structure-Based Design of N-Substituted 1-Hydroxy-4-sulfamoyl-2-naphthoates as Selective Inhibitors of the Mcl-1 Oncoprotein

PubMed Central

Lanning, Maryanna E.; Yu, Wenbo; Yap, Jeremy L.; Chauhan, Jay; Chen, Lijia; Whiting, Ellis; Pidugu, Lakshmi S.; Atkinson, Tyler; Bailey, Hala; Li, Willy; Roth, Braden M.; Hynicka, Lauren; Chesko, Kirsty; Toth, Eric A.; Shapiro, Paul; MacKerell, Alexander D.; Wilder, Paul T.; Fletcher, Steven

2016-01-01

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 and p3 pockets of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions with the p2 pockets dominate the affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 20-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukaemia. PMID:26985630

Prognostic significance of B-cell lymphoma 2 expression in acute leukemia: A systematic review and meta-analysis.

PubMed

Liu, Yanfeng; He, Pengcheng; Liu, Feng; Shi, Lili; Zhu, Huachao; Cheng, Xiaoyan; Zhao, Jing; Wang, Yuan; Zhang, Mei

2014-05-01

A number of studies have provided estimates of the correlation between B-cell lymphoma 2 (Bcl-2) expression and its clinical significance in acute leukemia (AL); however, the results have been heterogeneous. In order to clarify the prognostic significance of Bcl-2 status in patients with AL, a systematic review and meta-analysis of 5 published studies including a total of 665 subjects was performed. The reported frequency of Bcl-2 expression was 0-99.00%. Bcl-2-positive patients had a higher median white blood cell count compared to Bcl-2-negative patients. Additionally, Bcl-2-negative patients had >2-fold higher odds of achieving complete remission (CR) compared to Bcl-2-positive patients. The summary hazard ratio of Bcl-2 negativity/positivity for CR was 0.62 [95% confidence interval: 0.53-0.81, P<0.001]. Although this meta-analysis was based on data abstracted from observational studies, our results may justify the use of risk-adapted therapeutic strategies for AL according to the Bcl-2 expression status.

Follow-up care of young childhood cancer survivors: attendance and parental involvement.

PubMed

Vetsch, Janine; Rueegg, Corina S; Mader, Luzius; Bergstraesser, Eva; Rischewski, Johannes; Kuehni, Claudia E; Michel, Gisela

2016-07-01

Despite recommendations, only a proportion of long-term childhood cancer survivors attend follow-up care. We aimed to (1) describe the follow-up attendance of young survivors aged 11-17 years; (2) describe the parental involvement in follow-up, and (3) investigate predictors of follow-up attendance and parental involvement. As part of the Swiss Childhood Cancer Survivor Study, a follow-up questionnaire was sent to parents of childhood cancer survivors aged 11-17 years. We assessed follow-up attendance of the child, parents' involvement in follow-up, illness perception (Brief IPQ), and sociodemographic data. Clinical data was available from the Swiss Childhood Cancer Registry. Of 309 eligible parents, 189 responded (67 %; mean time since diagnosis 11.3 years, range 6.8-17.2) and 75 % (n = 141) reported that their child still attended follow-up. Of these, 83 % (n = 117) reported ≥1 visit per year and 17 % (n = 23) reported <1 visit every year. Most survivors saw pediatric oncologists (n = 111; 79 % of 141), followed by endocrinologists (n = 24, 17 %) and general practitioners (n = 22, 16 %). Most parents (92 %) reported being involved in follow-up (n = 130). In multivariable and Cox regression analyses, longer time since diagnosis (p = 0.025) and lower perceived treatment control (assessed by IPQ4: how much parents thought follow-up can help with late effects; p = 0.009) were associated with non-attendance. Parents' overall information needs was significantly associated with parental involvement in the multivariable model (p = 0.041). Educating survivors and their parents on the importance and effectiveness of follow-up care might increase attendance in the longer term.

Ameliorative Effects of Endurance Exercise with Two Different Intensities on Nandrolone Decanoate-Induced Neurodegeneration in Rats: Involving Redox and Apoptotic Systems.

PubMed

Joukar, Siyavash; Vahidi, Reza; Farsinejad, Alireza; Asadi-Shekaari, Majid; Shahouzehi, Beydolah

2017-07-01

Despite the importance of this issue, less has been paid to the influence of exercise on the neural side effects of anabolic androgenic steroids and mechanisms. We investigated the effects of two levels of endurance exercise on neurodegeneration side effects of nandrolone. The study period was 8 weeks. Wistar rats were divided into nine groups including the control (CTL) group, mild exercise (mEx) group, and vehicle (Arach) group which received arachis oil intramuscularly, nandrolone (Nan) group which received nandrolone decanoate 5 mg/kg two times weekly, mEx+Arach group which treated with arachis oil along with mild exercise, mEx+Nan group which treated with nandrolone along with mild exercise, severe exercise (sEx) group, sEx+Arach, and sEx+Nan groups. Finally, brain samples were taken for histopathological, biochemical, and western blot analysis. Nandrolone significantly decreased the intact cells of the hippocampus, total antioxidant capacity (TAC) (P < 0.05 versus CTL and Arach groups), TAC to malondialdehyde ratio (TAC/MDA), and Bcl-2. Nandrolone increased the Bax/Bcl-2 ratio of the brain tissue (P < 0.01 versus CTL and Arach groups). Combination of mild exercise and nandrolone rescued the intact cells to some extent, and this effect was associated with the improvement of Bcl-2 level and Bax/Bcl-2 ratio of brain tissue. Combination of severe exercise and nandrolone rescued the intact cells and improved the TAC, TAC/MDA, and Bax/Bcl-2 ratios. The findings suggest that low- and high-intensity endurance exercise decreased the risk of neurodegeneration effect of nandrolone in the hippocampus of rats. This effect can be explained by the regulation of the redox system and cell homeostasis.

Mapping of the bcl-2 oncogene on mouse chromosome 1.

PubMed

Mock, B A; Givol, D; D'Hoostelaere, L A; Huppi, K; Seldin, M F; Gurfinkel, N; Unger, T; Potter, M; Mushinski, J F

1988-01-01

Two bcl-2 alleles have been identified in inbred strains of mice by restriction fragment length polymorphism (RFLP). Analysis of a bcl-2 RFLP in a series of bilineal congenic strains (C.D2), developed as a tool for chromosomal mapping studies, revealed linkage of bcl-2 to the Idh-1/Pep-3 region of murine chromosome 1. The co-segregation of bcl-2 alleles with allelic forms of two other chromosome 1 loci, Ren-1,2 and Spna-1, in a set of back-cross progeny, positions bcl-2 7.8 cM centromeric from Ren-1,2.

Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells.

PubMed

Jiang, Kai; Wang, Wei; Jin, Xin; Wang, Zhaoyang; Ji, Zhiwei; Meng, Guanmin

2015-06-01

Silibinin, derived from the milk thistle plant (Silybum marianum), has anticancer and chemopreventive properties. Silibinin has been reported to inhibit the growth of various types of cancer cells. However, the mechanisms by which silibinin exerts an anticancer effect are poorly defined. The present study aimed to investigate whether silibinin-induced cell death might be attributed to autophagy and the underlying mechanisms in human MCF7 breast cancer cells. Our results showed that silibinin-induced cell death was greatly abrogated by two specific autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin-A1 (Baf-A1). In addition, silibinin triggered the conversion of light chain 3 (LC3)-I to LC3-II, promoted the upregulation of Atg12-Atg5 formation, increased Beclin-1 expression, and decreased the Bcl-2 level. Moreover, we noted elevated reactive oxygen species (ROS) generation, concomitant with the dissipation of mitochondrial transmembrane potential (ΔΨm) and a drastic decline in ATP levels following silibinin treatment, which were effectively prevented by the antioxidants, N-acetylcysteine and ascorbic acid. Silibinin stimulated the expression of Bcl-2 adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-death Bcl-2 family member, and silencing of BNIP3 greatly inhibited silibinin-induced cell death, decreased ROS production, and sustained ΔΨm and ATP levels. Taken together, these findings revealed that silibinin induced autophagic cell death through ROS-dependent mitochondrial dysfunction and ATP depletion involving BNIP3 in MCF7 cells.

Hsp70 suppresses apoptosis of BRL cells by regulating the expression of Bcl-2, cytochrome C, and caspase 8/3.

PubMed

Kong, Fanzhi; Wang, Hui; Guo, Jingru; Peng, Mengling; Ji, Hong; Yang, Huanmin; Liu, Binrun; Wang, Jianfa; Zhang, Xu; Li, Shize

2016-05-01

During cold stress, liver cells undergo apoptotic injury as a result of oxidative stress. Heat shock 70 kDa protein (Hsp70) is a protein involved in modulating a variety of physiological processes, including stress responses, proliferation, and apoptosis. In addition, Hsp70 regulates apoptotic signaling pathways in different manners, promoting or suppressing apoptosis. In this study, we investigated the effects of Hsp70 overexpression on hydrogen peroxide (H2O2)-induced apoptosis of Buffalo rat liver (BRL) cells and the underlying mechanisms of these effects. Our results show that in comparison with the control group, Hsp70 overexpression displayed increased protein levels of Bcl-2, and decreased cytochrome c (Cyt c), cleaved caspase 3, and cleaved caspase 8, but no apparent differences were found in levels of Bax. Furthermore, Hsp70 overexpression significantly suppresses the amount of apoptotic cells. Such findings indicate that overexpression of Hsp70 inhibits H2O2-mediated activation of caspase 8 and caspase 3, upregulates the expression of Bcl-2 which is a known anti-apoptotic protein, and decreases the release of Cyt c from the mitochondria into the cytoplasm, collectively decreasing cell apoptosis.

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

Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

PubMed Central

Lee, Woo Sang; Woo, Eun Young; Kwon, Junhye; Park, Myung-Jin; Lee, Jae-Seon; Han, Young-Hoon; Bae, In Hwa

2013-01-01

Bcl-w a pro-survival member of the Bcl-2 protein family, is expressed in a variety of cancer types, including gastric and colorectal adenocarcinomas, as well as glioblastoma multiforme (GBM), the most common and lethal brain tumor type. Previously, we demonstrated that Bcl-w is upregulated in gastric cancer cells, particularly those displaying infiltrative morphology. These reports propose that Bcl-w is strongly associated with aggressive characteristic, such as invasive or mesenchymal phenotype of GBM. However, there is no information from studies of the role of Bcl-w in GBM. In the current study, we showed that Bcl-w is upregulated in human glioblastoma multiforme (WHO grade IV) tissues, compared with normal and glioma (WHO grade III) tissues. Bcl-w promotes the mesenchymal traits of glioblastoma cells by inducing vimentin expression via activation of transcription factors, β-catenin, Twist1 and Snail in glioblastoma U251 cells. Moreover, Bcl-w induces invasiveness by promoting MMP-2 and FAK activation via the PI3K-p-Akt-p-GSK3β-β-catenin pathway. We further confirmed that Bcl-w has the capacity to induce invasiveness in several human cancer cell lines. In particular, Bcl-w-stimulated β-catenin is translocated into the nucleus as a transcription factor and promotes the expression of target genes, such as mesenchymal markers or MMPs, thereby increasing mesenchymal traits and invasiveness. Our findings collectively indicate that Bcl-w functions as a positive regulator of invasiveness by inducing mesenchymal changes and that trigger their aggressiveness of glioblastoma cells. PMID:23826359

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.

Pharmacokinetics of the B-Cell Lymphoma 2 (Bcl-2) Inhibitor Venetoclax in Female Subjects with Systemic Lupus Erythematosus.

PubMed

Minocha, Mukul; Zeng, Jiewei; Medema, Jeroen K; Othman, Ahmed A

2018-01-15

Venetoclax is an oral selective Bcl-2 inhibitor approved for the treatment of patients with chronic lymphocytic leukemia with 17p deletion. Mechanistic and preclinical evidence warranted evaluation of venetoclax for the treatment of systemic lupus erythematosus (SLE). This work characterized the pharmacokinetics of venetoclax in female subjects with SLE. Single (10-500 mg) and multiple (30-600 mg) escalating doses of venetoclax or matching placebo were evaluated using randomized, double-blind, placebo-controlled designs (6 active and 2 placebo per dose with 73 unique SLE patients enrolled, 25 of whom enrolled twice). The multiple-dose evaluation consisted of two cycles, each with once-daily dosing for 7 days followed by a 21-day washout. Non-compartmental and population pharmacokinetic analyses of venetoclax serial plasma concentrations were conducted. Venetoclax exhibited approximately dose-proportional exposures, with peak concentrations observed 4-8 h post-dose. Venetoclax steady-state exposures were achieved by day 4 of dosing, and the median area under the plasma concentration-time curve (AUC) accumulation ratio ranged from 1.1 to 1.5. A two-compartment model with first-order absorption and elimination described venetoclax pharmacokinetics. The estimates (95% bootstrap confidence interval) for venetoclax apparent clearance, central and peripheral volumes of distribution, intercompartmental clearance, absorption rate constant, and lag time were 16.3 L/h (14.6-17.9), 37 L (26-57), 122 L (98-183), 3.7 L/h (2.6-5.0), 0.13 h -1 (0.11-0.17), and 1.6 h (1.6-1.7), respectively. The population estimate for venetoclax terminal-phase elimination half-life was approximately 28 h. In female subjects with SLE, venetoclax displayed pharmacokinetic characteristics consistent with previous observations in subjects with hematologic malignancies. CLINICALTRIALS. NCT01686555.

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

The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

PubMed Central

Kang, CongBao; Bharatham, Nagakumar; Chia, Joel; Mu, Yuguang; Baek, Kwanghee; Yoon, Ho Sup

2012-01-01

Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2. PMID:23272207

Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

PubMed

Monaco, Giovanni; Decrock, Elke; Nuyts, Koen; Wagner, Larry E; Luyten, Tomas; Strelkov, Sergei V; Missiaen, Ludwig; De Borggraeve, Wim M; Leybaert, Luc; Yule, David I; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

2013-01-01

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca(2+)-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+) release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

Expression of BCL-2 in liver grafts after adenoviral transfer improves survival following prolonged ischemia and reperfusion in rat liver transplantation.

PubMed

Kienle, K; Rentsch, M; Müller, T; Engelhard, N; Vogel, M; Jauch, K W; Beham, A

2005-01-01

Apoptosis represents a crucial mechanism of ischemia-reperfusion injury after liver transplantation. Bcl-2 may inhibit apoptosis. This study investigates the effect on ischemia/reperfusion injury and survival after rat liver transplantation of adenoviral bcl-2 transfer into donor livers. A nonreplicative adenovirus, expressing bcl-2 under control of a tetracyclin-inducible promoter (adv TetOn bcl-2) was used to treat male Lewis rats in combination with a second adenovirus transferring the TetOn repressor protein under control of a cytomegalovirus promoter (advCMVRep). Virus induction was achieved by addition of doxycyclin to the drinking water. Controls were pretreated with a control adenovirus (advCMV GFP) or with doxycycline. Liver transplantations were performed after 16-hour graft storage. Bcl-2 expression was evaluated by Western blot and immunohistology. Survival was monitored for 7 days, and tissue specimens were collected at 24 hours and 7 days post reperfusion. After pretreatment with advTetOn bcl-2/adv CMVRep, intrahepatic bcl-2 expression was evident at 24 hours and 7 days but was absent among controls. Bcl-2 expression was detected in hepatocytes and, to a high degree, in sinusoidal lining cells. TUNEL-positive sinusoidal lining cells were strikingly reduced after bcl-2 transfer (0.1 +/- 0.3 cells/hpf, mean +/- SD) compared to control virus (4.8 +/- 2.3) or doxycyclin-treated grafts (1.3 +/- 0.2); P < .05. After bcl-2 treatment, survival after transplantation was 100%, whereas it was 50% in both control groups (P = .035). The study shows the feasibility of transient, doxycyclin-controlled adenoviral gene transfer in a transplantation model. Bcl-2 expression increased survival after ischemia/reperfusion in rat liver transplantation, potentially through protection of sinusoidal lining cells.

Sporoderm-Broken Spores of Ganoderma lucidum Inhibit the Growth of Lung Cancer: Involvement of the Akt/mTOR Signaling Pathway.

PubMed

Chen, Yali; Lv, Jing; Li, Kun; Xu, Jing; Li, Mingyan; Zhang, Wen; Pang, Xiufeng

2016-10-01

The sporoderm-broken spores of Ganoderma lucidum (SBGS) and their extracts exhibited a wide range of biological activities. In the present study, we prepare ethanol/ethanol extract (E/E-SBGS) and ethanol/aqueous extract (E/A-SBGS) from SBGS and examine their antitumor activities against human lung cancer. Our results showed that E/E-SBGS, not E/A-SBGS, inhibited the survival and migration of lung cancer cells in a dose-dependent manner. E/E-SBGS arrested cell cycle at G2/M phase and triggered apoptosis by decreasing the expression and activity of cell cycle regulators, cyclin B1 and cdc2, as well as anti-apoptotic proteins, Bcl-2 and Bcl-xl. Consequently, colony formation of lung cancer cells was markedly blocked by E/E-SBGS at subtoxic concentrations. Oral administration of both E/E-SBGS and SBGS significantly suppressed tumor volume and tumor weight without gross toxicity in mice. Mechanism study showed that E/E-SBGS dose-dependently suppressed the activation of Akt, the mammalian target of rapamycin (mTOR) and their downstream molecules S6 kinase and 4E-BP1 in treated tumor cells. Taken together, these results indicate that the ethanol extract of sporoderm-broken spores of G. lucidum suppresses the growth of human lung cancer, at least in part, through inhibition of the Akt/mTOR signaling pathway, suggesting its potential role in cancer treatments.

BOK promotes chemical-induced hepatocarcinogenesis in mice.

PubMed

Rabachini, Tatiana; Fernandez-Marrero, Yuniel; Montani, Matteo; Loforese, Giulio; Sladky, Valentina; He, Zhaoyue; Bachmann, Daniel; Wicki, Simone; Villunger, Andreas; Stroka, Deborah; Kaufmann, Thomas

2018-03-01

BCL-2-related ovarian killer (BOK) is a conserved and widely expressed BCL-2 family member with sequence homology to pro-apoptotic BAX and BAK, but with poorly understood pathophysiological function. Since several members of the BCL-2 family are critically involved in the regulation of hepatocellular apoptosis and carcinogenesis we aimed to establish whether loss of BOK affects diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Short-term exposure to DEN lead to upregulation of BOK mRNA and protein in the liver. Of note, induction of CHOP and the pro-apoptotic BH3-only proteins PUMA and BIM by DEN was strongly reduced in the absence of BOK. Accordingly, Bok -/- mice were significantly protected from DEN-induced acute hepatocellular apoptosis and associated inflammation. As a consequence, Bok -/- animals were partially protected against chemical-induced hepatocarcinogenesis showing fewer and, surprisingly, also smaller tumors than WT controls. Gene expression profiling revealed that downregulation of BOK results in upregulation of genes involved in cell cycle arrest. Bok -/- hepatocellular carcinoma (HCC) displayed higher expression levels of the cyclin kinase inhibitors p19 INK4d and p21 cip1 . Accordingly, hepatocellular carcinoma in Bok -/- animals, BOK-deficient human HCC cell lines, as well as non-transformed cells, showed significantly less proliferation than BOK-proficient controls. We conclude that BOK is induced by DEN, contributes to DEN-induced hepatocellular apoptosis and resulting hepatocarcinogenesis. In line with its previously reported predominant localization at the endoplasmic reticulum, our findings support a role of BOK that links the cell cycle and cell death machineries upstream of mitochondrial damage.

INVESTIGATING THE INFLUENCE OF LONG RANGE TRANSPORT ON MERCURY DEPOSITION IN SOUTH FLORIDA

EPA Science Inventory

In 1999, the US EPA National Exposure Research Laboratory (NERL) through a Regional Applied Research Effort initiated a study to evaluate the magnitude of long-range transport of mercury through the marine free troposphere to South Florida via aircraft measurements (200 to 11,5...

Dexamethasone modifies mitomycin C-triggered interleukin-8 secretion in isolated human Tenon's capsule fibroblasts.

PubMed

Ho, Wei-Ting; Chen, Tsan-Chi; Chou, San-Fang; Chang, Shu-Wen

2014-07-01

Intraoperative mitomycin C (MMC) is widely used to prevent pterygium recurrence and glaucoma filtering bleb failure, but it has been shown to induce corneal inflammation and cell death. Postoperative dexamethasone (DEX) is advocated to reduce MMC-related inflammation and cell death in corneal fibroblasts. Nevertheless, its long-term regulation mechanism in Tenon's capsule remains to be explored. The purpose of this study was to investigate how DEX modifies MMC's effects in human Tenon's capsule fibroblasts (HTFs). HTFs isolated from the pterygium surgical patients (n = 6) were treated with MMC at 0, 0.1, 0.2, and 0.4 mg/ml for 5 min and incubated in DEX at 10 μM for 0, 1, 2, and 3 days. Recombinant interleukin-8 (IL-8) was used to verify the effect of MMC-related IL-8 secretion. Cell proliferation of all the treated cells was analyzed by WST-1 assay. The amount of IL-8 secretion in HTFs was determined by enzyme-linked immunosorbent assay. Immunoblotting assay was used to analyze the expression of peroxisome-proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma-extra large (Bcl-xL). Our results revealed that MMC significantly reduced the HTF cell proliferation rate. Additionally, MMC significantly upregulated IL-8 secretion in HTFs concentration-dependently. At 3 days post treatment (dpt), 5-min exposures to 0.1, 0.2, and 0.4 mg/ml MMC resulted in 1.4-fold (p = 0.012), 1.6-fold (p = 0.012), and 2.5-fold (p = 0.001) increases of IL-8 secretion. In contrast, DEX reversed the MMC-retarded cell proliferation rate (p = 0.036) and repressed MMC-related IL-8 secretion by 33.5% at 3 dpt (p = 0.003). Addition of recombinant IL-8 noticeably suppressed HTF cell proliferation in a concentration-dependent manner. DEX stimulated upregulation of both PPARγ and Bcl-xL at 1 dpt in normal HTFs and at 2 dpt in MMC-treated HTFs. PPARγ silencing reduced expression of PPARγ and Bcl-xL, but enhanced IL-8 secretion (p < 0.001). On the other hand, Bcl-xL silencing enhanced IL-8 secretion (p < 0.001), but did not affect PPARγ expression. These revealed that IL-8 secretion in HTFs is modulated by PPARγ-dependent Bcl-xL signaling. We conclude that DEX reversed the MMC-inhibited HTF cell proliferation via diminishing the MMC-induced IL-8 secretion, which resulted from a late-phase upregulation of the PPARγ and Bcl-xL. These long-term effects suggest a beneficial postoperative DEX treatment following intraoperative MMC application. Copyright © 2014 Elsevier Ltd. All rights reserved.

Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts.

PubMed

Némati, Fariba; de Montrion, Catherine; Lang, Guillaume; Kraus-Berthier, Laurence; Carita, Guillaume; Sastre-Garau, Xavier; Berniard, Aurélie; Vallerand, David; Geneste, Olivier; de Plater, Ludmilla; Pierré, Alain; Lockhart, Brian; Desjardins, Laurence; Piperno-Neumann, Sophie; Depil, Stéphane; Decaudin, Didier

2014-01-01

Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

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.

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 in non-small cell lung cancer cell lines.

Knockdown of long noncoding RNA 00152 (LINC00152) inhibits human retinoblastoma progression.

PubMed

Li, Songhe; Wen, Dacheng; Che, Songtian; Cui, Zhihua; Sun, Yabin; Ren, Hua; Hao, Jilong

2018-01-01

A growing body of evidence supports the involvement of long noncoding RNA 00152 (LINC00152) in the progression and metastasis of multiple cancers. However, the exact roles of LINC00152 in the progression of human retinoblastoma (RB) remain unknown. We explored the expression and biological function of human RB. The expression level of LINC00152 in RB tissues and cells was analyzed using quantitative real-time PCR. The function of LINC00152 was determined using a series of in vitro assays. In vivo, a nude mouse model was established to analyze the function of LINC00152. Gene and protein expressions were detected using quantitative real-time PCR and Western blot assays, respectively. The expression of LINC00152 mRNA was upregulated in RB tissues and cell lines. Knockdown of LINC00152 significantly inhibited cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and caspase-3 and caspase-8 activities in vitro, as well as suppressing tumorigenesis in vivo. We identified several genes related to proliferation, apoptosis, and invasion including Ki-67, Bcl-2, and MMP-9 that were transcriptionally inactivated by LINC00152. Taken together, these data implicate LINC00152 as a therapeutic target in RB.

Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

PubMed Central

Liang, Qiming; Chang, Brian; Lee, Patrick; Brulois, Kevin F.; Ge, Jianning; Shi, Mude; Rodgers, Mary A.; Feng, Pinghui; Oh, Byung-Ha; Liang, Chengyu

2015-01-01

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication. PMID:25740994

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.

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

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

Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang, E-mail: zhuanghu475000@sina.com

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP responsemore » element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced by ASP.« less

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

Long-term outcome of percutaneous alcohol embolization combined with percutaneous vertebroplasty in aggressive vertebral hemangiomas with epidural extension.

PubMed

Premat, Kévin; Clarençon, Frédéric; Cormier, Évelyne; Mahtout, Jugurtha; Bonaccorsi, Raphaël; Degos, Vincent; Chiras, Jacques

2017-07-01

To evaluate, on a long-term basis, the safety and effectiveness of percutaneous alcohol embolization (PAE) combined with percutaneous vertebroplasty (PVP) as a sole treatment for aggressive vertebral haemangiomas (AVHs) with epidural extension. From 1996 to 2015, 26 consecutive patients (15 women [58%] and 11 men; mean age 51.8 years [range: 19-75 years]) underwent PAE combined with PVP (performed at day 15) for the treatment of 27 AVHs with epidural extension. Clinical outcome was evaluated with a mean delay of 88.3 ± 53.3 months (range: 22-217 months). The primary endpoint was pain relief evaluated with a visual analogue scale (VAS). Pre-procedure mean VAS score was 7.23 ± 1.3 and significantly improved at last follow-up (m = 3.11 ± 1.9; p < 0.001). Ten patients (38.5%) remained asymptomatic. Eighty-eight percent of the patients with neurosensory disorders had complete regression of these symptoms. Two of the three patients with motor deficit did not show any improvement. No major complication was recorded. PAE combined with PVP is a minimally invasive safe and effective therapeutic approach for AVH with epidural involvement, even on long-term clinical outcome. This technique appears mainly effective for pain and neurosensory symptoms, but seems less effective for motor deficit relief. • Combination of PAE with PVP is a safe technique. • PAE combined with PVP is an effective treatment for sensory symptoms. • This strategy seems less effective in patients with motor deficits.

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

The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, 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 Central

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.

2011-01-01

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-XL, 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-XL 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-XL, 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-XL/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-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612. PMID:21148306

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.

Childhood nodal marginal zone lymphoma with unusual clinicopathologic and cytogenetic features for the pediatric variant: a case report.

PubMed

Aqil, Barina; Merritt, Brian Y; Elghetany, M Tarek; Kamdar, Kala Y; Lu, Xinyan Y; Curry, Choladda V

2015-01-01

Nodal marginal zone lymphoma (NMZL) is a B-cell lymphoma that shares morphologic and immunophenotypic features with extranodal and splenic marginal zone lymphomas but lacks extranodal or splenic involvement at presentation. NMZL occurs mostly in adults with no sex predilection, at advanced stage (III or IV), with frequent relapses and a high incidence of tumoral genetic abnormalities including trisomies 3 and 18 and gain of 7q. Pediatric NMZL, however, is a rare but distinct variant of NMZL with characteristic features including male predominance, asymptomatic and localized (stage I) disease, low relapse rates with excellent outcomes, and a lower incidence of essentially similar genetic aberrations compared to adult NMZL. Here we describe a unique case of childhood NMZL with unusual clinicopathologic features for the pediatric variant including generalized lymphadenopathy, high-stage disease with persistence after therapy, unusual immunophenotype (CD5, CD23, and BCL6 positive), and unique chromosomal abnormalities including monosomy 20 and add(10)(p11.2).

Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione

PubMed Central

Wilkins, Heather M.; Marquardt, Kristin; Lash, Lawrence H.; Linseman, Daniel A.

2011-01-01

Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS)1 and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 is resident in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH) which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic, HA14-1, induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was co-immunoprecipitated with GSH following chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by pre-incubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. Following co-transfection of CHO cells, Bcl-2 was co-immunoprecipitated with OGC and this novel interaction was significantly enhanced by glutathione monoethylester (GSH-MEE). Similarly, recombinant Bcl-2 interacted with recombinant OGC in the presence of GSH. Bcl-2 and OGC co-transfection in CHO cells significantly increased the mitochondrial glutathione pool. Finally, the ability of Bcl-2 to protect CHO cells from apoptosis induced by hydrogen peroxide was significantly attenuated by the OGC inhibitor phenylsuccinate. These data suggest that GSH binding by Bcl-2 enhances its affinity for the OGC. Bcl-2 and OGC appear to act in a coordinated manner to increase the mitochondrial glutathione pool and enhance resistance of cells to oxidative stress. We conclude that regulation of mitochondrial glutathione transport is a principal mechanism by which Bcl-2 suppresses MOS. PMID:22115789

Nupr1/Chop signal axis is involved in mitochondrion-related endothelial cell apoptosis induced by methamphetamine

PubMed Central

Cai, D; Huang, E; Luo, B; Yang, Y; Zhang, F; Liu, C; Lin, Z; Xie, W-B; Wang, H

2016-01-01

Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1–Chop/P53–PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1–Chop/P53–PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial cell apoptosis and may be a potential therapeutic target for METH-caused cardiovascular toxicity. Future studies using knockout animal models are warranted to substantiate the present findings. PMID:27031958

Differences in the cytogenetic alteration profiles of diffuse large B-cell lymphoma among Chinese and American patients.

PubMed

Chen, Yan; Dave, Bhavana J; Zhu, Xiongzeng; Chan, Wing C; Iqbal, Javeed; Sanger, Warren G; Fu, Kai

2013-05-01

To study the similarities and differences of cytogenetic alterations in diffuse large B-cell lymphoma (DLBCL) between Asian and Caucasian patients, we compared the cytogenetic profiles of Chinese and American DLBCL cases by analyzing conventional karyotypes and select fluorescence in situ hybridization (FISH) findings. We used interphase FISH analyses to determine the incidence of the t(14;18) and BCL6 and MYC rearrangements. Immunohistochemical analysis was used to categorize the lymphomas into the germinal center B-cell-like (GCB) or non-GCB-DLBCL subtypes, according to the Hans algorithm. Our data suggested that Chinese patients had cytogenetic profiles for GCB-DLBCL that differed from those of their American counterparts; specifically, the Chinese GCB patients exhibited greater frequencies of BCL6 rearrangements and gains of 1q and 11q but lower incidence of the t(14;18). Non-GCB-DLBCL in both the Chinese and American patients was characterized by recurrent gains of 3/3q and 18/18q. The incidences of both BCL6 rearrangement and t(14;18) were similar in Chinese and American non-GCB-DLBCL cases. Copyright © 2013 Elsevier Inc. All rights reserved.

[Effect of Magnolol on Proliferation and Apoptosis of HL-60 Cells and Its Molecular Mechanism].

PubMed

Fang, Ke; Yuan, Xiao-Fen; Liao, Qiong; Zhang, Zhi-Yong; Song, Guan-Hua; Guo, Qiang; Ren, Xia; Jiang, Guo-Sheng

2016-04-01

To investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism. MTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol (5, 10, 20, 40, 80 and 160 µg/ml). The morphological changes of HL-60 cells were examined by light microscopy, and DAPI staining was performed to observe the nuclear morphology of HL-60 cells. The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining. RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2. Western blot was performed to detect the protein expression of caspase family. The magnolol inhibited HL-60 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time- dependent manner (P < 0.05). HL-60 cells became small, even apoptotic bodies appeared after treatment with magnolol. In addition, nuclear condensation or fragmentation could be observed, which is the typical morphological features of apoptosis. When HL-60 cells were treated with 40 µg/ml of magnolol for 24 h, the ratio of early apoptotic cells reached to (11.7 ± 2.4) %, which was significant different from control (1.4 ± 1.1) % (P < 0.05). RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX, whereas down-regulated the expression of BCL-2. Western blot results showed that the cleavages of caspase-3, -8 and -9 were significantly enhanced by magnolol. The magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through up-regulation of BAX, down-regulation of BCL-2 and the activation of caspases.

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.

Hydrogen peroxide-induced apoptosis in human gingival fibroblasts

PubMed Central

Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

2015-01-01

In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria. PMID:26884825

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

PubMed

Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

2011-10-28

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

PubMed

Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

2009-08-17

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

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.

ABCG2 is a direct transcriptional target of hedgehog signaling and involved in stroma-induced drug tolerance in diffuse large B-cell lymphoma.

PubMed

Singh, R R; Kunkalla, K; Qu, C; Schlette, E; Neelapu, S S; Samaniego, F; Vega, F

2011-12-08

Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by the development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of antiapoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. Previously, our studies showed that high expression of adenosine triphosphate-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease- and failure-free survival. In this study, we have implicated activated hedgehog (Hh) signaling pathway as a key factor behind high ABCG2 expression in DLBCL through direct upregulation of ABCG2 gene transcription. We have identified a single binding site for GLI transcription factors in the ABCG2 promoter and established its functionality using luciferase reporter, site-directed mutagenesis and chromatin-immunoprecipitation assays. Furthermore, in DLBCL tumor samples, significantly high ABCG2 and GLI1 levels were found in DLBCL tumors with lymph node involvement in comparison with DLBCL tumor cells collected from pleural and/or peritoneal effusions. This suggests a role for the stromal microenvironment in maintaining high levels of ABCG2 and GLI1. Accordingly, in vitro co-culture of DLBCL cells with HS-5 stromal cells increased ABCG2 mRNA and protein levels by paracrine activation of Hh signaling. In addition to ABCG2, co-culture of DLBCL cells with HS-5 cells also resulted in increase expression of the antiapoptotic proteins BCL2, BCL-xL and BCL2A1 and in induced chemotolerance to doxorubicin and methotrexate, drugs routinely used for the treatment of DLBCL. Similarly, activation of Hh signaling in DLBCL cell lines with recombinant Shh N-terminal peptide resulted in increased expression of BCL2 and ABCG2 associated with increased chemotolerance. Finally, functional inhibition of ABCG2 drug efflux activity with fumitremorgin C or inhibition of Hh signaling with cyclopamine-KAAD abrogated the stroma-induced chemotolerance suggesting that targeting ABCG2 and Hh signaling may have therapeutic value in overcoming chemoresistance in DLBCL.

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.

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 protects the tumor cells from other types of therapy. However, it is possible that cells expressing very high levels of the anti-apoptotic proteins might still be resistant to PDT. The data suggest that PDT with a non-vascular-targeting photosensitizer might be effective in a combination treatment in which Bcl-2 and Bcl-xL are first photodamaged before delivery of a second agent.

N-myc downstream-regulated gene 1 promotes oxaliplatin-triggered apoptosis in colorectal cancer cells via enhancing the ubiquitination of Bcl-2.

PubMed

Yang, Xiao; Zhu, Fan; Yu, Chaoran; Lu, Jiaoyang; Zhang, Luyang; Lv, Yanfeng; Sun, Jing; Zheng, Minhua

2017-07-18

N-myc downstream-regulated gene1 (NDRG1) has been identified as a potent tumor suppressor gene. The molecular mechanisms of anti-tumor activity of NDRG1 involve its suppressive effects on a variety of tumorigenic signaling pathways. The purpose of this study was to investigate the role of NDRG1 in the apoptosis of colorectal cancer (CRC) cells. We first collected the clinical data of locally advanced rectal cancer (LARC) patients receiving oxaliplatin-based neoadjuvant chemotherapy in our medical center. Correlation analysis revealed that NDRG1 positively associated with the downstaging rates and prognosis of patients. Then, the effects of over-expression and depletion of NDRG1 gene on apoptosis of colorectal cancer were tested in vitro and in vivo. NDRG1 over-expression promoted apoptosis in colorectal cancer cells whereas depletion of NDRG1 resulted in resistance to oxaliplatin treatment. Furthermore, we observed that Bcl-2, a major anti-apoptotic protein, was regulated by NDRG1 at post-transcriptional level. By binding Protein kinase Cα (PKCα), a classical regulating factor of Bcl-2, NDRG1 enhanced the ubiquitination and degradation of Bcl-2, thus promoting apoptosis in CRC cells. In addition, NDRG1 inhibited tumor growth and promoted apoptosis in mouse xenograft model. In conclusion,NDRG1 promotes oxaliplatin-triggered apoptosis in colorectal cancer. Therefore, colorectal cancer patients can be stratified by the expression level of NDRG1. NDRG1-positive patients may benefit from oxaliplatin-containing chemotherapy regimens whereas those with negative NDRG1 expression should avoid the usage of this cytotoxic drug.

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.

The Selective Progesterone Receptor Modulator CDB4124 Inhibits Proliferation and Induces Apoptosis in Uterine Leiomyoma Cells

PubMed Central

Luo, Xia; Yin, Ping; Coon V., John S.; Cheng, You-Hong; Wiehle, Ronald D.; Bulun, Serdar E.

2009-01-01

Objective To evaluate the effects of selective progesterone receptor modulator CDB4124 on cell proliferation and apoptosis in cultured human uterine leiomyoma smooth muscle (LSM) cells and control myometrial smooth muscle (MSM) cells in matched uteri. Design Laboratory research. Setting Academic medical center. Patient(s) Premenopausal women (n=12) undergoing hysterectomy for leiomyoma-related symptoms. Intervention(s) Treatment of primary LSM and MSM cells with CDB4124 (10-8-10-6M) or vehicle for 24, 48 or 72 hours. Main Outcome Measure(s) Western blot for protein expression of proliferating cell nuclear antigen (PCNA), cleaved poly-adenosine 5’-diphosphate-ribose polymerase (PARP), Bcl-2 and Krüppel-like transcription factor 11 (KLF11); MTT assay to evaluate viable cell numbers; and real-time polymerase chain reaction to quantify mRNA levels. Result(s) Treatment with CDB4124 significantly decreased levels of the proliferation marker PCNA, the number of viable LSM cells, and the anti-apoptotic protein Bcl-2. On the other hand, treatment with CDB4124 increased levels of the apoptosis marker cleaved PARP and the tumor suppressor KLF11 in a dose- and time-dependent manner in LSM cells. In matched MSM cells, however, CDB4124 did not affect cell proliferation or apoptosis. Conclusion(s) CDB4124 selectively inhibits proliferation and induces apoptosis in LSM but not in MSM cells. PMID:20056218

Assessment of apoptosis in oesophageal carcinoma preoperatively treated by chemotherapy and radiotherapy.

PubMed

Moreira, L F; Naomoto, Y; Hamada, M; Kamikawa, Y; Orita, K

1995-01-01

Apoptosis, programmed cell death, was immunohistochemically determined in 55 samples of oesophageal squamous cell carcinoma using the BM1 Mab. Sections from patients not treated (group 1, n = 12) or preoperatively treated by chemotherapy (group 2, n = 11), radiation (group 3, n = 13) or both (group 4, n = 8), and 11 additional cases of high-grade dysplasia or early cancer were examined. Most of the apoptotic cells were BM1-positive and checked by TUNEL proved to be nick end positive. They accounted for 7 (11%), 19 (29%), 21 (32%) and 26 (38%) cells per field in those 4 groups respectively. Chemotherapy and/or radiation significantly increased the number of apoptotic cells as compared to controls (p = 0.029 and p = 0.029, respectively). To assess the implications of the oncogene expression in the apoptotic pathway, additional section stained with bcl2 and p53 were negative for bcl2 and were positive for p53 in 16 samples (37%). Overall, positive cases for p53 mutation showed a significantly decreased incidence of apoptotic cells (p = 0.03). These results suggest that in situ assessment of apoptotic response better correlates to the apoptosis induced by radiation than that by chemotherapy, that abnormalities of the p53 protein decrease the apoptotic response in oesophageal carcinoma, and that immunohistochemical analysis of p53 protein helps to determine the sensitivity to these anticancer agents.

Radiation dose effect of DNA repair-related gene expression in mouse white blood cells.

PubMed

Li, Ming-juan; Wang, Wei-wei; Chen, Shi-wei; Shen, Qian; Min, Rui

2011-10-01

The aim of this study was to screen molecular biomarkers for biodosimetry from DNA repair-related gene expression profiles. Mice were subjected to whole-body exposure with 60Co gamma rays with a dose range of 0-8 Gy at a dose rate of 0.80 Gy/min. RNA was extracted from the peripheral blood of irradiated mice at 4, 8, 12, 24 and 48hrs post-irradiation. The mRNA transcriptional changes of 11 genes related to DNA damage and repair were detected using real-time quantitative polymerase chain reaction (RT-PCR). Of the 11 genes examined, CDKN1A (cyclin-dependent kinase inhibitor 1A or p21, Cip1) and ATM (ataxia telangiectasia mutated) expression levels were found to be heavily up- and down-regulated, respectively, with exposure dose increasing at different post-irradiation times. RAD50 (RAD50 homolog), PLK3 (polo-like kinase 3), GADD45A (growth arrest and DNA damage-inducible, alpha), DDB2 (damage-specific DNA-binding protein 2), BBC3 (BCL2-binding component 3) and IER5 (immediate early response 5) gene expression levels were found to undergo significant oscillating changes over a broad dose range of 2-8 Gy at post-exposure time points observed. Three of the genes were found not to change within the observed exposure dose and post-radiation time ranges. The results of this study add to the biodosimetry with biomarker data pool and will be helpful for constructing appropriate gene expression biomarker systems to evaluate radiation exposure doses.

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.

Inhibition of Bcl-2 Sensitizes Mitochondrial Permeability Transition Pore (MPTP) Opening in Ischemia-Damaged Mitochondria

PubMed Central

Chen, Qun; Xu, Haishan; Xu, Aijun; Ross, Thomas; Bowler, Elizabeth; Hu, Ying; Lesnefsky, Edward J.

2015-01-01

Background Mitochondria are critical to cardiac injury during reperfusion as a result of damage sustained during ischemia, including the loss of bcl-2. We asked if bcl-2 depletion not only leads to selective permeation of the outer mitochondrial membrane (MOMP) favoring cytochrome c release and programmed cell death, but also favors opening of the mitochondrial permeability transition pore (MPTP). An increase in MPTP susceptibility would support a role for bcl-2 depletion mediated cell death in the calcium overload setting of early reperfusion via MPTP as well as later in reperfusion via MOMP as myocardial calcium content normalizes. Methods Calcium retention capacity (CRC) was used to reflect the sensitivity of the MPTP opening in isolated cardiac mitochondria. To study the relationship between bcl-2 inhibition and MPTP opening, mitochondria were incubated with a bcl-2 inhibitor (HA14-1) and CRC measured. The contribution of preserved bcl-2 content to MPTP opening following ischemia-reperfusion was explored using transgenic bcl-2 overexpressed mice. Results CRC was decreased in mitochondria following reperfusion compared to ischemia alone, indicating that reperfusion further sensitizes to MPTP opening. Incubation of ischemia-damaged mitochondria with increasing HA14-1concentrations increased calcium-stimulated MPTP opening, supporting that functional inhibition of bcl-2 during simulated reperfusion favors MPTP opening. Moreover, HA14-1 sensitivity was increased by ischemia compared to non-ischemic controls. Overexpression of bcl-2 attenuated MPTP opening in following ischemia-reperfusion. HA14-1 inhibition also increased the permeability of the outer membrane in the absence of exogenous calcium, indicating that bcl-2 inhibition favors MOMP when calcium is low. Conclusions The depletion and functional inhibition of bcl-2 contributes to cardiac injury by increasing susceptibility to MPTP opening in high calcium environments and MOMP in the absence of calcium overload. Thus, ischemia-damaged mitochondria with decreased bcl-2 content are susceptible to MPTP opening in early reperfusion and MOMP later in reperfusion when cytosolic calcium has normalized. PMID:25756500

[Effects of acupotomy, electroacupuncture or round-sharp acupuncture needle interventions on expression of Bcl-2, Bax, Caspase-3 proteins of rectus femoris in rabbits with knee ostarthritis].

PubMed

Huang, Yi-Ran; Jin, Ying-Li; Li, Na; Lei, Lin-Dan; Yu, Fei; Li, Yu-Bo; Tao, Lin; Zhang, Qian; Guo, Chang-Qing

2014-04-01

To observe the effect of acupotomy, electroacupuncture (EA) or round-sharp acupuncture needle intervention on the expression of Bcl-2,Bax and Caspase-3 proteins in the rectus femoris in rabbits with knee ostarthritis (KOA), so as to explore their mechanisms underlying improvement of braking-induced joint damage from the cellular apoptosis. Forty-five New Zealand rabbits were equally and randomized into control group, model group, acupotomy (AP) group, EA group and round-sharp acupuncture needle (RSAN) group (n = 9 in each group). The knee-joint injury model was established by fixing the left knee joint in extention position with plaster bandage. EA (2 Hz/100 Hz, 3 mA, 20 min each time) was applied to the left "Yanglingquan" (GB 34)- "Yinlingquan" (SP 9) and left "Neixiyan" (EX-LE 4)- "Waixiyan"(ST 35) for rabbits in the EA group. The EA treatment was given once daily, 3 times a week, 3 weeks in total. For rabbits of the AP group, a needle-knife was held to insert into the front edge of the midpoint, the starting point and the stopping point of the left medial collateral ligamen, lateral collateral ligament and the patellar ligament of the knee to make a loosening manipulation for 5 times in a session of treatment, once a week, 3 times altogether. For rabbits of the RSAN group, a round-sharp needle was performed in the same way to the needle-knife including the stimulation point, the manipulation method and treatment sessions. At the end of the experiment, the left rectus femoris was taken out for detecting the expression of Bcl-2, Bax and Caspase-3 proteins with Western blot. In comparison with the control group, the passive range of motion (PROM) level was significantly decreased 4, 8 and 12 weeks after modeling (P < 0.01), and the expression levels of Bax and Caspase-3 proteins in the rectus femoris were considerably upregulated in the model group (P < 0.05), while the ratio of Bcl-2/Bax was notably down-regulated (P < 0.05) in the model group. Compared with the model group, the PROM level at week 12 after modeling in the AP, EA and RSAN groups were significantly increased (P < 0.01); while Bax and Caspase-3 expression levels in both AP and RSAN groups were considerably downregulated (P < 0.05). No significant differences were found among the five groups in Bcl-2 expression levels (P > 0.05), and between the EA and model groups in Bax and Caspase-3 expression levels and the ratio of Bcl-2/Bax (P > 0.05). AP, RSAN and EA interventions are effective in improving the knee-joint motion range in KOA rabbits, and this effect of both AP and RSAN is closely associated with their actions in lowering the expression of Bax and Caspase-3 proteins of the rectus femoris and in raising ratio of Bcl-2/Bax protein (reducing muscular cellular apoptosis). The mechanism of EA intervention in improving PROM may be different.

PROGNOSTIC FACTORS FOR PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMAS TREATED WITH HIGH-DOSE METHOTREXATE-BASED CHEMO-RADIOTHERAPY

PubMed Central

Nagane, Motoo; Lee, Jeunghun; Shishido-Hara, Yukiko; Suzuki, Kaori; Shimizu, Saki; Umino, Michiru; Kobayashi, Keiichi; Shiokawa, Yoshiaki

2014-01-01

BACKGROUND: Chemotherapy with high-dose methotrexate (HD-MTX) followed by whole brain radiotherapy (WBRT) is a conventional approach to treat primary central nervous system lymphomas (PCNSL), but some tumors relapse early leading to unfavorable outcome. Several biomarkers have been identified as prognostic factors in PCNSL, however, the correlation of both clinical factors including those related to MTX metabolism and B-cell differentiation and oncogenic biomarkers with response to and outcome by therapy is yet unclear. METHODS: We investigated 32 immunocompetent patients (19 males, 13 females) with PCNSL (all diffuse large B-cell type) treated with HD-MTX based therapy with or without WBRT since 2000 in our institution. Paraffin-embedded formalin-fixed tumor tissue sections were stained immunohistochemically with antibodies against following factors: B-cell differentiation markers (CD10, Bcl-6, Mum-1, CD138); MTX metabolism-related (MRP family, LRP, DHFR); cell cycle-related (p27KIP1, MIB-1); drug resistance-related (MGMT, MLH1, MSH2, MSH6, PMS2); and oncogenes (Myc, Bcl-2). Correlation between positivity of these factors and clinical outcomes were evaluated using logrank test and cox regression analysis. RESULTS: Among these factors, complete response to HD-MTX was significantly associated with longer progression-free survival (PFS)(P = 0.0012), while Bcl-6 expression as well as histological subtype (non-germinal center B-cell, non-GCB) was closely correlated with shorter PFS. Age (>60) (P = 0.006) and MSH2 expression (P = 0.017) were found to be better predictor for overall survival (OS), but in multivariate analysis, they were no longer significant. Other factors involved in MTX metabolism, DNA repair enzymes, and oncogenes did not affect outcome. CONCLUSIONS: Non-GCB subtype and Bcl-6 expression may be associated with worse outcome in patients with PCNSL treated with HD-MTX, while MTX-metabolism related factors did not influence prognosis. Further investigation is needed to assess Bcl-6 as a potential prognostic factor in PCNSL. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Structural Factors and Mechanisms Underlying the Improved Photodynamic Cell Killing with Silicon Phthalocyanine Photosensitizers Directed to Lysosomes Versus Mitochondria

PubMed Central

Rodriguez, Myriam E.; Zhang, Ping; Azizuddin, Kashif; Delos Santos, Grace B.; Chiu, Song-mao; Xue, Liang-yan; Berlin, Jeffery C.; Peng, Xinzhan; Wu, Hongqiao; Lam, Minh; Nieminen, Anna-Liisa; Kenney, Malcolm E.; Oleinick, Nancy L.

2012-01-01

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway. PMID:19508642

BAG3-mediated Mcl-1 stabilization contributes to drug resistance via interaction with USP9X in ovarian cancer.

PubMed

Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

2016-07-01

Paclitaxel in combination with carboplatin improves survival among patients with susceptible ovarian cancers, but no strategy has been established against resistant ovarian cancers. BAG3 (Bcl-2-associated athanogene 3) is one of six BAG family proteins, which are involved in such cellular processes as proliferation, migration and apoptosis. In addition, expression of BAG3 with Mcl-1, a Bcl-2 family protein, reportedly associates with resistance to chemotherapy. Our aim in this study was to evaluate the functional role of BAG3 and Mcl-1 in ovarian cancer chemoresistance and explore possible new targets for treatment. We found that combined expression of BAG3 and Mcl-1 was significantly associated with a poor prognosis in ovarian cancer patients. In vitro, BAG3 knockdown in ES2 clear ovarian cancer cells significantly increased the efficacy of paclitaxel in combination with the Mcl-1 antagonist MIM1, with or without the Bcl-2 family antagonist ABT737. Moreover, BAG3 was found to positively regulate Mcl-1 levels by binding to and inhibiting USP9X. Our data show that BAG3 and Mcl-1 are key mediators of resistance to chemotherapy in ovarian cancer. In BAG3 knockdown ES2 clear ovarian cancer cells, combination with ABT737 and MIM1 enhanced the efficacy of paclitaxel. These results suggest that inhibiting BAG3 in addition to anti-apoptotic Bcl-2 family proteins may be a useful therapeutic strategy for the treatment of chemoresistant ovarian cancers.

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

Small Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy

PubMed Central

Han, Bingshe; Park, Dongkyoo; Li, Rui; Xie, Maohua; Owonikoko, Taofeek K.; Zhang, Guojing; Sica, Gabriel L.; Ding, Chunyong; Zhou, Jia; Magis, Andrew T.; Chen, Zhuo G.; Shin, Dong M.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Curran, Walter J.; Deng, Xingming

2015-01-01

SUMMARY The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small molecule Bcl2-BH4 domain-antagonist (BDA-366) that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition up-regulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome. PMID:26004684

Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury.

PubMed

Imahashi, Kenichi; Schneider, Michael D; Steenbergen, Charles; Murphy, Elizabeth

2004-10-01

The antiapoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival after ischemia, or whether it also affects metabolic functions of mitochondria during ischemia. Hearts from mice overexpressing human Bcl-2 and from their wild-type littermates (WT) were subjected to 24 minutes of global ischemia followed by reperfusion. During ischemia, the decrease in pH(i) and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts (P<0.05). The reduced acidification during ischemia was dependent on the activity of mitochondrial F1F0-ATPase. In the presence of oligomycin (Oligo), an F1F0-ATPase inhibitor, the decrease in pH(i) was attenuated in WT hearts, but in Bcl-2 hearts, Oligo had no additional effect on pH(i) during ischemia. Likewise, addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These data are consistent with Bcl-2-mediated inhibition of consumption of glycolytic ATP. Furthermore, mitochondria from Bcl-2 hearts have a reduced rate of consumption of ATP on uncoupler addition. This could be accomplished by limiting ATP entry into the mitochondria through the voltage-dependent anion channel, and/or the adenine nucleotide transporter, or by direct inhibition of the F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and voltage-dependent anion channel during ischemia. These data indicate that Bcl-2 modulation of metabolism contributes to cardioprotection.

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

PubMed

Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2014-01-01

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

Induction of p53-mediated apoptosis in splenocytes and thymocytes of C57BL/6 mice exposed to perfluorooctane sulfonate (PFOS)

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

Dong, Guang-Hui, E-mail: ghdong@mail.cmu.edu.cn; Wang, Jing; Zhang, Ying-Hua

2012-10-15

Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant found in human and wildlife tissues. It has been reported that PFOS can cause atrophy of the immune organs and apoptosis of immunocytes in rodents. However, the mechanism behind such cause is still unclear. To understand the model of cell death and its mechanism on lymphoid cells in vivo, we conducted a dose/response experiment in which 4 groups of male adult C57BL/6 mice (12 mice per group) were dosed daily by oral gavage with PFOS at 0, 0.0167, 0.0833, or 0.8333 mg/kg/day, yielding targeted Total Administered Dose (TAD) of 0, 1, 5,more » or 50 mg PFOS/kg, respectively, over 60 days. The results showed that spleen and thymus weight were significantly reduced in the highest PFOS-dose-group (TAD 50 mg PFOS/kg) compared to the control group, whereas liver weight was significantly increased. We analyzed the cell death via apoptosis with an annexin-V/propidium iodide assay by flow cytometry, and observed that both the percentage of apoptosis and the expression of the pro-apoptotic proteins p53 in splenocytes and thymocytes increased in a dose-related manner after PFOS treatment. We also observed that PFOS induced p53-dependent apoptosis through the cooperation between the Bcl-xl down regulation without changing the Bcl-2 and Bax expression. The down regulation of Bcl-xl was strongly indicating mitochondrial involvement in apoptosis. It is confirmed by the release of cytochrome c and activation of caspase-3. All of these findings establish an important role of p53 and mitochondrial function in PFOS induced toxic environment in the host. -- Highlights: ► PFOS immunotoxicity is caused by induction of apoptosis via the p53 activation. ► PFOS exposure can induce down regulation of Bcl-xl. ► Mitochondria are involved in PFOS-induced apoptosis. ► PFOS exposure can cause the release of cytochrome c and activation of caspase-3.« less

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.

[Influence of chronic alcohol treatment on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes in the mouse brain: Role of the C1473G polymorphism in the gene encoding tryptophan hydroxylase 2].

PubMed

Bazovkina, D V; Tsybko, A S; Filimonova, E A; Ilchibaeva, T V; Naumenko, V S

2016-01-01

Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment.

SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

PubMed

Jia, Zhuqing; Wang, Jiaji; Shi, Qiong; Liu, Siyu; Wang, Weiping; Tian, Yuyao; Lu, Qin; Chen, Ping; Ma, Kangtao; Zhou, Chunyan

2016-02-01

Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

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.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

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

Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui

2015-04-01

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressedmore » c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.« less

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.

Blue light-induced apoptosis of human promyelocytic leukemia cells via the mitochondrial-mediated signaling pathway.

PubMed

Zhuang, Jianjian; Liu, Yange; Yuan, Qingxia; Liu, Junsong; Liu, Yan; Li, Hongdong; Wang, Di

2018-05-01

Acute promyelocytic leukemia is frequently associated with dizziness, fever, nausea, hematochezia and anemia. Blue light, or light with wavelengths of 400-480 nm, transmits high levels of energy. The aim of the present study was to determine the pro-apoptotic effects of blue light (wavelength, 456 nm; radiation power, 0.25 mW/cm 2 ) and the underlying mechanisms in a human promyelocytic leukemia cell line (HL60). Blue light reduced the viability and enhanced the mortality of HL60 cells in a time-dependent manner. Exposure to blue light for 24 h caused depolarization of the mitochondrial membrane potential and the overproduction of reactive oxygen species in HL60 cells. In a nude mouse model, 9-day exposure to blue light markedly suppressed the growth of HL60-xenografted tumors; however, it had no effect on hepatic and renal tissues. In addition, blue light abrogated the expression of B-cell lymphoma (Bcl)-2 and Bcl extra-long, while enhancing the levels of Bcl-2-associated X protein, cytochrome c , and cleaved caspases-3 and -9 in tumor tissues. The results suggested that the pro-apoptotic effects of blue light in human promyelocytic leukemia cells may be associated with the mitochondrial apoptosis signaling pathway.

Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma.

PubMed

Tam, Constantine S; Anderson, Mary Ann; Pott, Christiane; Agarwal, Rishu; Handunnetti, Sasanka; Hicks, Rodney J; Burbury, Kate; Turner, Gillian; Di Iulio, Juliana; Bressel, Mathias; Westerman, David; Lade, Stephen; Dreyling, Martin; Dawson, Sarah-Jane; Dawson, Mark A; Seymour, John F; Roberts, Andrew W

2018-03-29

Both the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax are active as monotherapy in the treatment of mantle-cell lymphoma. Complete response rates of 21% have been observed for each agent when administered as long-term continuous therapy. Preclinical models predict synergy in combination. We conducted a single-group, phase 2 study of daily oral ibrutinib and venetoclax in patients, as compared with historical controls. Patients commenced ibrutinib monotherapy at a dose of 560 mg per day. After 4 weeks, venetoclax was added in stepwise, weekly increasing doses to 400 mg per day. Both drugs were continued until progression or an unacceptable level of adverse events. The primary end point was the rate of complete response at week 16. Minimal residual disease (MRD) was assessed by flow cytometry in bone marrow and by allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) in blood. The study included 24 patients with relapsed or refractory mantle-cell lymphoma (23 patients) or previously untreated mantle-cell lymphoma (1 patient). Patients were 47 to 81 years of age, and the number of previous treatments ranged from none to six. Half the patients had aberrations of TP53, and 75% had a high-risk prognostic score. The complete response rate according to computed tomography at week 16 was 42%, which was higher than the historical result of 9% at this time point with ibrutinib monotherapy (P<0.001). The rate of complete response as assessed by positron-emission tomography was 62% at week 16 and 71% overall. MRD clearance was confirmed by flow cytometry in 67% of the patients and by ASO-PCR in 38%. In a time-to-event analysis, 78% of the patients with a response were estimated to have an ongoing response at 15 months. The tumor lysis syndrome occurred in 2 patients. Common side effects were generally low grade and included diarrhea (in 83% of the patients), fatigue (in 75%), and nausea or vomiting (in 71%). In this study involving historical controls, dual targeting of BTK and BCL2 with ibrutinib and venetoclax was consistent with improved outcomes in patients with mantle-cell lymphoma who had been predicted to have poor outcomes with current therapy. (Funded by Janssen and others; AIM ClinicalTrials.gov number, NCT02471391 .).