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MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis  [PDF]
Jae-Hong Kim, Jeehyeon Bae
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0079626
Abstract: MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.
Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells  [cached]
Palve Vinayak C,Teni Tanuja R
Radiation Oncology , 2012, DOI: 10.1186/1748-717x-7-135
Abstract: Background Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy. Methods The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR). Results Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells, suggesting a synergistic effect of the Mcl-1L siRNA with IR on radiosensitivity. Interestingly, during the development of radioresistant sublines using FIR, high expression of Mcl-1L was observed. Conclusion Our studies suggest that Mcl-1L isoform has an important role in the survival and radioresistance of OSCC and may be a promising therapeutic target in oral cancers.
Serine 162, an Essential Residue for the Mitochondrial Localization, Stability and Anti-Apoptotic Function of Mcl-1  [PDF]
Luke W. Thomas, Connie Lam, Richard E. Clark, Michael R. H. White, David G. Spiller, Robert J. Moots, Steven W. Edwards
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045088
Abstract: Mcl-1 is an anti-apoptotic member of the Bcl-2 family that plays a key role in normal development, but also in pathologies such as cancer. It has some unusual properties compared to other anti-apoptotic members of the Bcl-2 family, and its expression and function are dynamically regulated by a variety of post-transcriptional and post-translational processes. Of note, Mcl-1 protein has a very short half life, and its stability and function may be regulated by reversible phosphorylation. There is also evidence to suggest that it may be localized to different subcellular compartments. The aim of this work was to determine whether residues within the PEST region of Mcl-1 that may undergo reversible phosphorylation, also regulate its subcellular distribution. We show that EGFP:Mcl-1 localizes mainly to the mitochondria of HeLa cells, with some additional cytoplasmic and nuclear localization. The mutations, S64A, S64E, S121A, S159A, T163A and T163E did not significantly affect the localization of Mcl-1. However, mutation of Ser162 to the phospho-null residue, Alanine resulted in an essentially nuclear localization, with some cytoplasmic but no mitochondrial localization. This mutant Mcl-1 protein, S162A, showed significantly decreased stability and it decreased the ability to protect against Bak-induced apoptosis. These data identify a new molecular determinant of Mcl-1 function, localization and stability that may be important for understanding the role of this protein in disease.
Molecular Interactions of Prodiginines with the BH3 Domain of Anti-Apoptotic Bcl-2 Family Members  [PDF]
Ali Hosseini, Margarita Espona-Fiedler, Vanessa Soto-Cerrato, Roberto Quesada, Ricardo Pérez-Tomás, Victor Guallar
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057562
Abstract: Prodigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins.
The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites
Michael Lutter, Guy A Perkins, Xiaodong Wang
BMC Cell Biology , 2001, DOI: 10.1186/1471-2121-2-22
Abstract: We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBidG94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBidG94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBidG94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.Recent work has shown that the Bcl-2 family regulates mitochondrial homeostasis during apoptosis [1]. Pro-apoptotic members, including Bax, Bak, Bid, and Bim, promote the release of death-inducing proteins, such as cytochrome c [2,3], smac [4], and endonuclease G [5], from mitochondria while anti-apoptotic members, such as Bcl-2 and Bcl-XL, inhibit this release. Following release into the cytosol, these death-inducing proteins promote apoptotic cell destruction through multiple pathways including caspase activation and nuclear DNA fragmentation.In addition to controlling the release of pro-death proteins, the Bcl-2 family also alters the function of mitochondria undergoing apoptosis. Dysfunction of vo
c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1
Mario Campone, Bélinda No?l, Cécile Couriaud, Morgan Grau, Yannis Guillemin, Fabien Gautier, Wilfried Gouraud, Catherine Charbonnel, Lo?c Campion, Pascal Jézéquel, Frédérique Braun, Benjamin Barré, Olivier Coqueret, Sophie Barillé-Nion, Philippe Juin
Molecular Cancer , 2011, DOI: 10.1186/1476-4598-10-110
Abstract: We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data.We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that na?ve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors.This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.Breast cancer is a heterogeneous disease, composed of distinct entities with differing underlying pathogenic processes. One such entity is the so-called HER2 subtype, which is characterized by amplification and/or overexpression of this member of the human epidermal growth factor receptor (HER) family. HER2 is an orphan receptor with intrinsic tyrosine kinase activity [1] whose activation results from the dynamic heterodimerization of HER receptors members [2]. This activates a large repertoire of transforming signaling molecules and pathways that are, to a great extent, shared by HER
Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family
Meng-Lay Lin, Jae-Hyun Park, Toshihiko Nishidate, Yusuke Nakamura, Toyomasa Katagiri
Breast Cancer Research , 2007, DOI: 10.1186/bcr1650
Abstract: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.Breast cancer is one of the leading causes of cancer death in women worldwide. According to a 2002 estimate, more than 1,100,000 patients were newly diagnosed with breast cancer, and approximately 4
The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax, modulate functional interactions with the anti-apoptotic protein Bcl-xL
Neha Parikh, Caroline Koshy, Vaigundan Dhayabaran, Lakshmi R Perumalsamy, R Sowdhamini, Apurva Sarin
BMC Cell Biology , 2007, DOI: 10.1186/1471-2121-8-16
Abstract: Deletion of 29 amino acids in the Bax N-terminus (Bax 30–192) caused constitutive accumulation at mitochondria and triggered high levels of cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30–105) abrogated mitochondrial localization but resulted in Bcl-xL regulated activation of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the α5-α6 helices/TMI domain (Bax 30–146) phenocopied Bax 30–192 as it restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not dependent on endogenous Bax-Bak. Inhibition of function and localization by Bcl-xL was restored in Bax 1–146, which included the TM1 domain. Regardless of regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated Bcl-xLand converged on caspase-9 dependent apoptosis consistent with mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence alignments of Bax and Bcl-xL indicated a sequence similarity between the α5–α6 helices of Bax and Bcl-xL. Alanine substitutions of three residues (T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct regulatory mechanisms.Collectively, the analysis of Bax deletion constructs indicates that the N-terminus drives conformational changes facilitating inhibition of cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as 'structural antagonists' for BH3-Bcl-xL interactions, with this function being regulated by the N-terminus in the intact protein.Bcl-2 family proteins are central to the regulation of mitochondrial integrity and cell death pathways that converge on this organelle [1,2]. This family comprises both pro-survival (Bcl-2, Bcl-xL, Mcl-1, Bcl-w) and pro-apoptotic (Bax, Bak, Bim, Bid, Bad) proteins. All members of this family are characterized by the presence of BH (Bcl-2 homology) domain(s) [3,4]. Bax is a multidomain pro-apoptotic member of the Bcl-2 family, characterized by the prese
Mcl-1 Is a Key Regulator of Apoptosis Resistance in Chlamydia trachomatis-Infected Cells  [PDF]
Krishnaraj Rajalingam, Manu Sharma, Christine Lohmann, Monique Oswald, Oliver Thieck, Christopher J. Froelich, Thomas Rudel
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003102
Abstract: Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.
The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity  [PDF]
Ryan C. Thompson, Iosif Vardinogiannis, Thomas D. Gilmore
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062822
Abstract: Background Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease and this variation can often be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi’s) as monotherapy or in combination with other agents. Methodology/Principal Findings We have used a variety of cell-based and molecular/biochemical assays to show that two pan-HDAC inhibitors, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Consistent with previous reports implicating the BCL-2 family in regulating HDACi-induced apoptosis, ectopic over-expression of anti-apoptotic proteins BCL-2 and BCL-XL or pro-apoptotic protein BIM in these cell lines conferred further resistance or sensitivity, respectively, to HDACi treatment. Additionally, BCL-2 family antgonist ABT-737 increased the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including one cell line (SUDHL6) that is resistant to vorinostat alone. Moreover, two variants of the HDACi-sensitive SUDHL4 cell line that have decreased sensitivity to vorinostat showed up-regulation of BCL-2 family anti-apoptotic proteins such as BCL-XL and MCL-1, as well as decreased sensitivity to ABT-737. These results suggest that the regulation and overall balance of anti- to pro-apoptotic BCL-2 family protein expression is important in defining the sensitivity of DLBCL to HDACi-induced apoptosis. However, the sensitivity of DLBCL cell lines to HDACi treatment does not correlate with expression of any individual BCL-2 family member. Conclusions/Significance These studies indicate that the sensitivity of DLBCL to treatment with HDACi’s is dependent on the complex regulation of BCL-2 family members and that BCL-2 antagonists may enhance the response of a subset of DLBCL patients to HDACi treatment.
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