DOCSLIB.ORG

Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol Published OnlineFirst March 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0909 Molecular Cancer Preclinical Development Therapeutics Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol Jenna L. Riffell1, Reiner U. Janicke€ 2, and Michel Roberge1 Abstract Microtubule-targeting cancer drugs such as paclitaxel block cell-cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells can spontaneously escape mitotic arrest and enter interphase without chromosome segregation by a process termed mitotic slippage that involves the degradation of cyclin B1 without mitotic checkpoint inactivation. Inducing mitotic slippage with chemicals causes cells to die after multiple rounds of DNA replication without cell division, which may enhance the antitumor activity of microtubule-targeting drugs. Here, we explore pathways leading to mitotic slippage by using SU6656 and geraldol, two recently identified chemical inducers of mitotic slippage. Mitotic slippage induced by SU6656 or geraldol was blocked by the proteasome inhibitor MG-132 and involved proteasome-dependent degradation of cyclin B1 and the mitotic checkpoint proteins budding uninhibited by benzimidazole related 1 (BubR1) and cell division cycle 20 (Cdc20) in T98G cells. Mitotic slippage and the degradation of BubR1 and Cdc20 were also inhibited by the caspase-3 and -7 inhibitor DEVD-CHO. MCF-7 cells lacking caspase-3 expression could not degrade BubR1 or undergo mitotic slippage in response to SU6656 or geraldol. Introduction of caspase-3 completely restored the ability of MCF-7 cells to degrade BubR1 and undergo mitotic slippage. However, lack of expression of caspase-3 did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy. Mol Cancer Ther; 10(5); 839–49. Ó2011 AACR. Introduction mosome separation when even 1 kinetochore is unat- tached. Exposure to drugs that interfere with microtubule During cell division, genetic integrity is maintained by dynamics, such as the taxanes (4) and the Vinca alkaloids ensuring that all chromosomes are attached to microtu- (5), similarly activates the mitotic checkpoint and arrests bules emanating from both poles of the mitotic spindle cells at mitosis, effectively preventing further prolifera- before segregation of sister chromatids begins (1). This tion. process is monitored by the mitotic checkpoint, which The mitotic checkpoint acts through inhibition of the prevents initiation of anaphase until every kinetochore is anaphase-promoting complex/cyclosome (APC/C; ref. attached and tension between kinetochores of paired 6), the E3 ubiquitin ligase (7) that, when activated by sister chromatids is sufficient, ensuring biorientation cofactors cell division cycle 20 (Cdc20) or Cdh1 (8), (2). To prevent aneuploidy and ensuing genetic defects polyubiquitylates the cyclin-dependent kinase 1 (Cdk1) leading to cell death or tumorigenesis (3), the mitotic cofactor cyclin B1 (7) and the separase regulator securin checkpoint must be sufficiently sensitive to delay chro- (9), targeting them for degradation by the proteasome. This results in inactivation of Cdk1, separation of sister chromatids, and exit from mitosis. The key components Authors' Affiliations: 1Department of Biochemistry and Molecular Biol- ogy, University of British Columbia, Vancouver, British Columbia, Canada; of the mitotic checkpoint are budding uninhibited by and 2Laboratory for Molecular Radiooncology, Clinic and Policlinic for benzimidazole related 1 (BubR1), budding uninhibited Radiation Therapy and Radiooncology, Heinrich Heine Universitat€ by benzimidazole 3 (Bub3), and Cdc20, which form a Dusseldorf,€ Dusseldorf,€ Germany mitotic checkpoint complex (MCC; ref. 10). This complex Note: Supplementary data for this article are available at Molecular Cancer is the main inhibitor of APC/C activity, along with Therapeutics Online (http://mct.aacrjournals.org/). mitotic arrest dependent 2 (Mad2), which initially binds Corresponding Author: Michel Roberge, Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Cdc20 (11) and catalyzes its binding to BubR1 and sub- Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Phone: sequent formation of the MCC (12). Cdc20 is an activating 604-822-2304; Fax: 604-822-5227. E-mail: [email protected] cofactor of APC/C during mitosis (8); an active mitotic doi: 10.1158/1535-7163.MCT-10-0909 checkpoint inhibits APC/C through APC/C-dependent Ó2011 American Association for Cancer Research. polyubiquitylation of Cdc20 and subsequent degradation www.aacrjournals.org 839 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst March 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0909 Riffell et al. by the proteasome (12). BubR1 binds to and inhibits both Materials and Methods Cdc20 (13) and APC/C itself (14), acting as a pseudosub- strate inhibitor that, depending on acetylation status, can Cell culture and chemicals be actively degraded by APC/CCdc20 (15). The role of T98G cells, obtained from the American Type Culture Bub3 in the MCC is unclear, although in fission yeast it is Collection (ATCC; characterized by short tandem repeat involved in MCC localization (16). Other components of analysis) and used within 6 months of resuscitation, were the mitotic checkpoint include the kinases Bub1, mono- maintained in Dulbecco’s Modified Eagle’s Medium polar spindle 1 (Mps1), and Aurora B (2). (Invitrogen) supplemented with 10% FBS (Gibco). Caspases have well-characterized apoptotic functions, MCF-7 cell lines, obtained from the ATCC and stably but caspase-3 and caspase-7 have both recently been transfected with empty vector (pcDNA) or caspase-3 observed to play a role, yet to be defined, in mitotic (casp3), were maintained in RPMI (Invitrogen) supple- progression (17, 18). Their activities are tightly regulated mented with 10% FBS and 10 mmol/L HEPES, pH 7.3 and must be restrained during mitotic stress to prevent (Invitrogen). Paclitaxel was obtained from USB, SU6656, extensive cell death, most notably through survivin, and MG-132 from Sigma, geraldol from Chromadex, and which inhibits caspase activation during mitotic arrest cell-permeable DEVD-CHO from Enzo Life Sciences. and functions as part of the mitotic checkpoint machinery (19). Slippage induction assay Mitotic checkpoint activation during an unperturbed T98G cells at 75% confluency were treated with 30 mitosis provides sufficient time for microtubule attach- nmol/L paclitaxel, or MCF-7 cells were treated with 50 ment, preventing aneuploidy (20) and increasing cell nmol/L paclitaxel, for 20 hours at 37 C, and mitotic cells survival (21). However, long-term activation of the were harvested by shake-off, counted using a hemacyt- mitotic checkpoint during exposure to antimitotic ometer, seeded in a 96-well plate (PerkinElmer View- agents can be problematic because chromosome con- plate) at 5,000 cells per well, and treated with chemicals as densation hinders RNA transcription (22). With time, an indicated for 4 hours at 37 C. Unattached mitotic cells imbalance between new protein production and protein were then aspirated and discarded while attached, degradation may cause the levels of proteins essential to slipped cells were fixed in 3% paraformaldehyde maintain mitotic arrest to fall, triggering mitotic slip- (EMD) in PBS for 15 minutes at room temperature, and page. Also termed mitotic checkpoint adaptation, mito- stained with Hoechst 33342 (Invitrogen) in PBS for 10 tic slippage occurs when cells exit mitosis without minutes at room temperature. Five fields per well were chromosome segregation or cell division (20, 23) and counted by a Cellomics ArrayScan VTI automated fluor- results from slow APC/CCdc20- and proteasome-depen- escence imager (ThermoFisher) by using a 10Â objective. dent degradation of cyclin B1 in the presence of an Individual nuclei of slipped cells were detected and active mitotic checkpoint (24, 25). Cells that have under- counted using the Cellomics Target Activation Analysis gone mitotic slippage enter a G1-like state with decon- Program. In all figures, mitotic slippage was expressed as densed chromosomes that form multiple micronuclei a percentage of the cells seeded in each well (26). (23), allowing resumption of transcription and other cellular processes. Immunoblotting Our group and others have identified chemicals that Cells were washed in PBS and lysed for 5 minutes on stimulate mitotic slippage and observed that slipped ice in lysis buffer containing 20 mmol/L Tris-HCl cells typically undergo at least 1 round of DNA replica- (Fisher), pH 7.5, 150 mmol/L NaCl (Fisher), 1 mmol/L tion without subsequent cell division but that, even- EDTA (Sigma), 1 mmol/L EGTA (Sigma), 1% Triton tually, all cells that undergo mitotic slippage die (26– X-100 (LabChem Inc.), 2.5 mmol/L sodium pyropho- 30). Known chemical inducers of mitotic slippage sphate (Fisher), 1 mmol/L b-glycerol phosphate (Sigma), include CDK1 inhibitors (roscovitine, RO3066; ref. 1 mmol/L sodium orthovanadate (Sigma), and 1Â pro- 28), histone deacetylase complex inhibitors (SBHA, tease inhibitor cocktail (Roche). Lysates were spun at SAHA, sodium butyrate, trichostatin
Load more

Recommended publications
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • HMGB1 in Health and Disease R
    Donald and Barbara Zucker School of Medicine Journal Articles Academic Works 2014 HMGB1 in health and disease R. Kang R. C. Chen Q. H. Zhang W. Hou S. Wu See next page for additional authors Follow this and additional works at: https://academicworks.medicine.hofstra.edu/articles Part of the Emergency Medicine Commons Recommended Citation Kang R, Chen R, Zhang Q, Hou W, Wu S, Fan X, Yan Z, Sun X, Wang H, Tang D, . HMGB1 in health and disease. 2014 Jan 01; 40():Article 533 [ p.]. Available from: https://academicworks.medicine.hofstra.edu/articles/533. Free full text article. This Article is brought to you for free and open access by Donald and Barbara Zucker School of Medicine Academic Works. It has been accepted for inclusion in Journal Articles by an authorized administrator of Donald and Barbara Zucker School of Medicine Academic Works. Authors R. Kang, R. C. Chen, Q. H. Zhang, W. Hou, S. Wu, X. G. Fan, Z. W. Yan, X. F. Sun, H. C. Wang, D. L. Tang, and +8 additional authors This article is available at Donald and Barbara Zucker School of Medicine Academic Works: https://academicworks.medicine.hofstra.edu/articles/533 NIH Public Access Author Manuscript Mol Aspects Med. Author manuscript; available in PMC 2015 December 01. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Mol Aspects Med. 2014 December ; 0: 1–116. doi:10.1016/j.mam.2014.05.001. HMGB1 in Health and Disease Rui Kang1,*, Ruochan Chen1, Qiuhong Zhang1, Wen Hou1, Sha Wu1, Lizhi Cao2, Jin Huang3, Yan Yu2, Xue-gong Fan4, Zhengwen Yan1,5, Xiaofang Sun6, Haichao Wang7, Qingde Wang1, Allan Tsung1, Timothy R.
    [Show full text]
  • The Role of Caspase-2 in Regulating Cell Fate
    cells Review The Role of Caspase-2 in Regulating Cell Fate Vasanthy Vigneswara and Zubair Ahmed * Neuroscience and Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham B15 2TT, UK; [email protected] * Correspondence: [email protected] Received: 15 April 2020; Accepted: 12 May 2020; Published: 19 May 2020 Abstract: Caspase-2 is the most evolutionarily conserved member of the mammalian caspase family and has been implicated in both apoptotic and non-apoptotic signaling pathways, including tumor suppression, cell cycle regulation, and DNA repair. A myriad of signaling molecules is associated with the tight regulation of caspase-2 to mediate multiple cellular processes far beyond apoptotic cell death. This review provides a comprehensive overview of the literature pertaining to possible sophisticated molecular mechanisms underlying the multifaceted process of caspase-2 activation and to highlight its interplay between factors that promote or suppress apoptosis in a complicated regulatory network that determines the fate of a cell from its birth and throughout its life. Keywords: caspase-2; procaspase; apoptosis; splice variants; activation; intrinsic; extrinsic; neurons 1. Introduction Apoptosis, or programmed cell death (PCD), plays a pivotal role during embryonic development through to adulthood in multi-cellular organisms to eliminate excessive and potentially compromised cells under physiological conditions to maintain cellular homeostasis [1]. However, dysregulation of the apoptotic signaling pathway is implicated in a variety of pathological conditions. For example, excessive apoptosis can lead to neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, whilst insufficient apoptosis results in cancer and autoimmune disorders [2,3]. Apoptosis is mediated by two well-known classical signaling pathways, namely the extrinsic or death receptor-dependent pathway and the intrinsic or mitochondria-dependent pathway.
    [Show full text]
  • Separase Protease Activity Is Required for Cytokinesis in Addition
    bioRxiv preprint doi: https://doi.org/10.1101/069906; this version posted August 16, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Separase Protease Activity is Required for Cytokinesis in addition to Chromosome Segregation Xiaofei Bai1 and Joshua N. Bembenek1* 1Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, United States of America * Corresponding Author: Joshua N. Bembenek 1414 Cumberland Ave. C211 Walters Life Sciences Building Knoxville, TN 37996 (865)-974-4085 E-mail: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/069906; this version posted August 16, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Chromosomal segregation and cytokinesis are tightly regulated processes required for successful cell division. The cysteine protease separase cleaves a subunit of the cohesin complex to allow chromosome segregation at anaphase onset. Separase also regulates meiotic cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. Separase has non-proteolytic signaling functions in addition to its role in substrate cleavage, and its mechanism in exocytosis is unknown. We sought to determine whether separase regulates RAB-11 vesicle exocytosis through a proteolytic or non-proteolytic mechanism.
    [Show full text]
  • Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice
    The Journal of Neuroscience, November 3, 2004 • 24(44):9977–9984 • 9977 Development/Plasticity/Repair Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice Caroline Houde,1 Kathleen G. Banks,2 Nathalie Coulombe,3 Dita Rasper,3 Erich Grimm,3 Sophie Roy,1,4 Elizabeth M. Simpson,2 and Donald W. Nicholson1,4 1Biochemistry Department, McGill University, Montreal, Quebec H3G 1Y6, Canada, 2Centre for Molecular Medicine and Therapeutics, British Columbia Institute for Children’s and Women’s Health, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada, 3Merck Frosst Canada and Company, Pointe-Claire–Dorval, Quebec H9R 4P8, Canada, and 4Merck Research Laboratories, San Diego, California 92121 Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain- Ϫ Ϫ dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3 / ) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with Ϫ Ϫ Ϫ Ϫ lack of DNA fragmentation in 129-Casp3 / apoptotic precursor neurons, whereas B6-Casp3 / cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation.
    [Show full text]
  • Proteolytic Enzymes in Grass Pollen and Their Relationship to Allergenic Proteins
    Proteolytic Enzymes in Grass Pollen and their Relationship to Allergenic Proteins By Rohit G. Saldanha A thesis submitted in fulfilment of the requirements for the degree of Masters by Research Faculty of Medicine The University of New South Wales March 2005 TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF FIGURES 6 LIST OF TABLES 8 LIST OF TABLES 8 ABBREVIATIONS 8 ACKNOWLEDGEMENTS 11 PUBLISHED WORK FROM THIS THESIS 12 ABSTRACT 13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES 14 1.1 Defining Asthma and its Clinical Presentation 14 1.2 Inflammatory Responses in Asthma 15 1.2.1 The Early Phase Response 15 1.2.2 The Late Phase Reaction 16 1.3 Effects of Airway Inflammation 16 1.3.1 Respiratory Epithelium 16 1.3.2 Airway Remodelling 17 1.4 Classification of Asthma 18 1.4.1 Extrinsic Asthma 19 1.4.2 Intrinsic Asthma 19 1.5 Prevalence of Asthma 20 1.6 Immunological Sensitisation 22 1.7 Antigen Presentation and development of T cell Responses. 22 1.8 Factors Influencing T cell Activation Responses 25 1.8.1 Co-Stimulatory Interactions 25 1.8.2 Cognate Cellular Interactions 26 1.8.3 Soluble Pro-inflammatory Factors 26 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation 30 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES 33 1 2.1 The Role of Pollen Allergens in Asthma 33 2.2 Environmental Factors influencing Pollen Exposure 33 2.3 Classification of Pollen Sources 35 2.3.1 Taxonomy of Pollen Sources 35 2.3.2 Cross-Reactivity between different Pollen Allergens 40 2.4 Classification of Pollen Allergens 41 2.4.1
    [Show full text]
  • Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-Induced Apoptosis of Jurkat Cells HIROAKI SUITA, TAKAHISA SHINOMIYA and YUKITOSHI NAGAHARA
    ANTICANCER RESEARCH 37 : 1697-1704 (2017) doi:10.21873/anticanres.11501 Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells HIROAKI SUITA, TAKAHISA SHINOMIYA and YUKITOSHI NAGAHARA Division of Life Science and Engineering, School of Science and Engineering, Tokyo Denki University, Hatoyama, Japan Abstract. Background: Mitochondria are central to caspases (caspase-8 and -9) that activate effector caspases (1). apoptosis. However, apoptosis progression involving Caspases are constructed of a pro-domain, a large subunit, and mitochondria is not fully understood. A factor involved in a small subunit. Upon caspase activation, cleavage of the mitochondria-mediated apoptosis is 7A6 antigen. 7A6 caspase occurs. Caspase-6 and -7, which are effector caspases, localizes to mitochondria from the cytosol during apoptosis, each have a large subunit that is 20 kDa (p20) and a small which seems to involve ‘effector’ caspases. In this study, we subunit that is 10 kDa (p10), but caspase-3, which is also an investigated the precise role of effector caspases in 7A6 effector caspase, has a large subunit of 17 kDa and a small localization to mitochondria during apoptosis. Materials and subunit of 12 kDa (p12) (2-4). Moreover, the large and small Methods: Human T-cell lymphoma Jurkat cells were treated subunits of caspase-6 and -7 are connected by a linker, but with an antibody against FAS. 7A6 localization was analyzed caspase-3 does not have a linker (Figure 1). The difference by confocal laser scanning microscopy and flow cytometry. between effector and initiator caspases is that the pro-domain Caspases activation was determined by western blot of an initiator caspase is long and that the pro-domain of an analysis.
    [Show full text]
  • Executioner Caspase-3 and Caspase-7 Are Functionally Distinct Proteases
    Executioner caspase-3 and caspase-7 are functionally distinct proteases John G. Walsh, Sean P. Cullen, Clare Sheridan, Alexander U. Luthi,¨ Christopher Gerner*, and Seamus J. Martin† Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland Edited by Doug R. Green, St. Jude Children’s Research Hospital, Memphis, TN, and accepted by the Editorial Board July 8, 2008 (received for review August 15, 2007) Members of the caspase family of cysteine proteases play central CASP-3 and CASP-7 on the B6 background die immediately roles in coordinating the stereotypical events that occur during after birth because of defective heart development (9). The latter apoptosis. Because the major executioner caspases, caspase-3 and result suggests that there may be a degree of functional com- caspase-7, exhibit almost indistinguishable activity toward certain pensation in operation between caspase-3 and caspase-7, synthetic peptide substrates, this has led to the widespread view whereas the knockout phenotypes observed on the 129 back- that these proteases occupy functionally redundant roles within ground suggest that caspase-3 and caspase-7 serve distinct roles. the cell death machinery. However, the distinct phenotypes of mice However, a major problem in interpreting these data is that the deficient in either of these caspases, as well as mice deficient in relative expression levels of caspase-3 and caspase-7 may vary both, is at odds with this view. These distinct phenotypes could be dramatically between mouse strains, as well as within particular related to differences in the relative expression levels of caspase-3 tissues.
    [Show full text]
  • Developmental Requirements for Ubiquitin-Mediated Proteolysis
    REVIEW 773 Development 133, 773-784 doi:10.1242/dev.02276 Degrade to create: developmental requirements for ubiquitin-mediated proteolysis during early C. elegans embryogenesis Bruce Bowerman1 and Thimo Kurz2,* The ubiquitin protein conjugation system tags proteins with the the activity of cell division kinases, which must bind to cyclin small polypeptide ubiquitin. Most poly-ubiquitinated proteins proteins to be active (Morgan, 1997; Murray, 2004). Cyclins were are recognized and degraded by the proteasome, a large multi- so named because they accumulate to peak levels at specific times subunit protease. Ubiquitin-dependent protein degradation is during the cell cycle, thereby activating cell division kinases at used as a regulatory tool for many essential processes, the best appropriate moments. A regulatory role for ubiquitin in the cell cycle studied of which is eukaryotic cell cycle progression. More was discovered when it was shown that cyclins are targeted for rapid recently, genetic studies in C. elegans have identified multiple degradation during the cell cycle by poly-ubiquitination (Glotzer et roles for the ubiquitin system in early development, where al., 1991; Hershko et al., 1991). ubiquitin-dependent protein degradation governs such diverse Additional roles for ubiquitination emerged from studies of the events as passage through meiosis, cytoskeletal regulation and metaphase-to-anaphase transition during mitosis (Peters, 2002). cell fate determination. Early in mitosis, the duplicated chromosomes (sister chromatids) condense and are held together by a multi-protein cohesin complex. Introduction As the bipolar mitotic spindle forms, sister chromatid pairs become Ubiquitin-mediated proteolysis, discovered over 25 years ago captured by microtubules such that each spindle pole is attached to (Ciechanover et al., 1980; Hershko et al., 1980), covalently attaches only one sister.
    [Show full text]
  • Contribution of Calpain and Caspases to Cell Death in Cultured Monkey RPE Cells
    Biochemistry and Molecular Biology Contribution of Calpain and Caspases to Cell Death in Cultured Monkey RPE Cells Emi Nakajima,1,2 Katherine B. Hammond,1,2 Masayuki Hirata,1,2 Thomas R. Shearer,2 and Mitsuyoshi Azuma1,2 1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, Oregon, United States 2Department of Integrative Biosciences, Oregon Health & Science University, Portland, Oregon, United States Correspondence: Mitsuyoshi Azuma, PURPOSE. AMD is the leading cause of human vision loss after 65 years of age. Several Senju Pharmaceutical Corporation mechanisms have been proposed: (1) age-related failure of the choroidal vasculature leads to Limited, 4640 SW Macadam Avenue, loss of RPE; (2) RPE dysfunctions due to accumulation of phagocytized, but unreleased A2E Suite 200C, Portland, OR 97239, (N-retinylidene-N-retinylethanolamine); (3) zinc deficiency activation of calpain and caspase USA; proteases, leading to cell death. The purpose of the present study is to compare activation of [email protected]. calpain and caspase in monkey RPE cells cultured under hypoxia or with A2E. Submitted: June 1, 2017 Accepted: September 19, 2017 METHODS. Monkey primary RPE cells were cultured under hypoxic conditions in a Gaspak pouch or cultured with synthetic A2E. Immunoblotting was used to detect activation of Citation: Nakajima E, Hammond KB, calpain and caspase. Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were Hirata M, Shearer TR, Azuma M. Contribution of calpain and caspases used to confirm activation of the proteases. to cell death in cultured monkey RPE RESULTS. (1) Hypoxia and A2E each decreased viability of RPE cells in a time-dependent cells.
    [Show full text]
  • ^ P X R, for the PURPOSES of INFORMATION ONLY
    WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 6 : (11) International Publication Number: WO 98/49190 C07K 5/06, 5/08, 5/10 A l (43) International Publication Date: 5 November 1998 (05.11.98) (21) International Application Number: PCT/US98/08259 (74) Agents: BURKE, John, E. et al.; Cushman Darby & Cushman, Intellectual Property Group of Pillsbury Madison & Sutro, (22) International Filing Date: 24 April 1998 (24.04.98) 1100 New York Avenue, N.W., Washington, DC 20005 (US). (30) Priority Data: 60/044,819 25 April 1997 (25.04.97) US (81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, Not furnished 23 April 1998 (23.04.98) US BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, (71) Applicant (for all designated States except US): CORTECH, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, INC. [US/US]; 6850 North Broadway, Denver, CO 80221 TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO (US). patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (72) Inventors; and patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, (75) Inventors/Applicants(for US only): SPRUCE, Lyle, W. IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, [US/US]; 948 Camino Del Sol, Chula Vista, CA 91910 CG, Cl, CM, GA, GN, ML, MR, NE, SN, TD, TG).
    [Show full text]
  • Intertwined Functions of Separase and Caspase in Cell Division
    bioRxiv preprint doi: https://doi.org/10.1101/653584; this version posted May 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death Pan-Young Jeong1, Ashish Kumar1, Pradeep Joshi1, and Joel H. Rothman1* Affiliations: 1Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA. *Correspondence to: [email protected] Key words : C. elegans, chromosome separation, separase, caspase, programmed cell death 2 bioRxiv preprint doi: https://doi.org/10.1101/653584; this version posted May 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract: Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report that the C. elegans separase SEP-1, primarily known for its role in cell division, is required for apoptosis when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation and germline meiotic chromosome disjunction and the normal rate of embryonic development.
    [Show full text]