Doctoral dissertation
To be presented by permission of the Faculty of Medicine of the University of Kuopio
for public examination in Auditorium ML3, Medistudia building, University of Kuopio,
on Friday 11th December 2009, at 12 noon
Institute of Clinical MedicineDepartment of Clinical Microbiology
University of Kuopio
ULLA NUUTINEN
Mechanisms and Regulation of Apoptotic Pathways in Human
Follicular Lymphoma Cells
Aiming for Curative Treatment
JOKAKUOPIO 2009
KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 466KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 466
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Series Editors: Professor Raimo Sulkava, M.D., Ph.D. School of Public Health and Clinical Nutrition Professor Markku Tammi, M.D., Ph.D. Institute of Biomedicine, Department of Anatomy
Author´s address: Institute of Clinical Medicine Department of Clinical Microbiology University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND E-mail : [email protected] Supervisors: Professor Jukka Pelkonen, M.D., Ph.D. Institute of Clinical Medicine Department of Clinical Microbiology University of Kuopio
Docent Pekka Riikonen, M.D. Department of Pediatrics Kuopio University Hospital
Reviewers: Docent Juha Klefström, Ph.D. Cancer Cell Circuitry Laboratory, Biomedicum Helsinki University of Helsinki
Adjunct Professor Kaisa Heiskanen, Ph.D. Orion Corporation ORION PHARMA Turku
Opponent: Professor Lea Sistonen, Ph.D. Turku Centre for Biotechnology, Department of Biology Åbo Akademi University
ISBN 978-951-27-1366-0ISBN 978-951-27-1383-7 (PDF)ISSN 1235-0303
KopijyväKuopio 2009Finland
Nuutinen, Ulla. Mechanisms and Regulation of Apoptotic Pathways in Human FollicularLymphoma Cells – Aiming for Curative Treatment. Kuopio University Publications D. MedicalSciences 466. 2009. 94 p.ISBN 978-951-27-1366-0ISBN 978-951-27-1383-7 (PDF)ISSN 1235-0303
ABSTRACT
Follicular lymphoma (FL) is still considered to be an incurable disease. The aim of this thesis was tostudy mechanisms and regulation of intrinsic and extrinsic apoptotic pathways induced bydexamethasone (Dex) and TRAIL, respectively, in human FL cell lines. In addition, the effect of animportant FL microenvironmental signal, CD40 signaling, on TRAIL-, Dex- and doxorubicin-inducedapoptosis was evaluated.Glucocorticoids (GCs) are commonly used in the treatment of lymphoid malignancies. However, themolecular mechanisms and regulation of GC-induced apoptosis remain still largely unknown.Mitochondria were necessary for Dex-induced apoptosis since overexpression of Bcl-XL prevented Dex-induced apoptotic changes. Interestingly, dominant negative (DN) caspase-9 also prevented Dex-inducedapoptotic changes including the loss of mitochondrial membrane potential indicating that caspase-9controls mitochondrial changes. New protein synthesis was required and the kinetics of the apoptoticevents correlated with Dex-induced upregulation of the Bim. Interestingly, inhibition of glycogensynthase kinase-3 (GSK3) attenuated Dex-induced up-regulation of Bim and other apoptotic changesindicating that Dex-induced apoptosis is dependent on GSK3. Furthermore, inhibition of PI3-kinase-Aktpathway markedly enhanced and accelerated Dex-induced apoptosis already at the mitochondrial levelby causing translocation of Bad to mitochondria. Thus, these results indicate that inhibitors of PI3-kinase-Akt pathway might be combined with glucocorticoids to improve the treatment of FL.The extrinsic apoptotic pathway is activated through engagement of the cell surface death receptors bytheir ligands. TRAIL has emerged as the most promising for the death receptor targeted cancer therapydue to its remarkable feature of selectively inducing apoptosis in cancer cells without causing toxicityfor normal cells. Based on Bcl-XL overexpression studies we identified type I (mitochondrialindependent) and type II (mitochondria dependent) follicular lymphoma cell lines in response to TRAIL.Furthermore, a NF- B inhibitor, PDTC enabled TRAIL to activate type I apoptotic pathway in Bcl-XLoverexpressing type II cells cells. However, an inhibitor of IKK did not switch apoptosis to type Ipathway in type II cells, indicating that NF- B might not be responsible for the switch. The activation ofapoptosis independently of mitochondria could have great advantage to treatment of FL because mostcases of FL overexpress anti-apoptotic mitochondrial Bcl-2 protein.FL cells are malignant counterparts of germinal center (GC) B cells. Microenvironment of FL B cellshas an important role in the progression of FL and might also have an impact on the treatment of FL.CD40 is an important mediator of microenvironmental survival signals in GCs. Therefore, we studiedresponses of CD40 signaling on TRAIL-, Dex- and doxorubicin-induced apoptosis in three human FLcell lines. In two of the FL cell lines, CD40 protected cells from apoptosis and the protection wasentirely dependent on the activation of NF- B. In one of the FL cell lines, CD40 induced apoptosisitself. However, inhibition of NF- B itself induced apoptosis in all three FL cell lines. Therefore, ourresults indicate that inhibitors of NF- B or critical downstream anti-apoptotic targets of NF- or insome cases blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should beconsidered in the treatment of FL in order to prevent the protective effect of the microenvironment.
National Library of Medicine Classification: QU 375, QZ 267, WH 200, WH 525Medical Subject Headings: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Line,Tumor; Dexamethasone; Human; Lymphoma, Follicular; Mitochondria; NF-kappa B; Signal Transduction;TNF-Related Apoptosis-Inducing Ligand; 1-Phosphatidylinositol 3-Kinase
To my family
ACKNOWLEDGEMENTS
This work was carried out at the Department of Clinical Microbiology, Institute of ClinicalMedicine, University of Kuopio, during the years 2003-2009.
I wish to express my deepest gratitude to my principal supervisor, Professor Jukka Pelkonen, M.D.,Ph.D., for his guidance, patience, open mind for new ideas and encouragement, during these years.I am deeply grateful to Jukka for opening the doors of the fascinating world of science to me andbeing there when I needed help and support. I am also deeply grateful to my second supervisorDocent Pekka Riikonen, M.D., for affording the clinical view for this research project.
I wish to thank Docent Tuomas Virtanen, M.D., Ph.D., and Professor Jorma Ilonen M.D., Ph.D.,for their scientific support and interesting discussions.
I express my sincere gratitude to all my collaborators. I wish to thank Mine Eray M.D., Ph.D., forpriceless collaboration, especially for the establishment of the human follicular lymphoma cell linesused in this study. I am also very grateful to Docent Jarmo Wahlfors, Ph.D., Riikka Pellinen, Ph.D.,Tanja Hakkarainen, Ph.D., and Katja Häkkinen, M.Sc., for their fruitful collaboration and help withviral transductions.
I owe my warmest thanks to all the present and former colleagues in the Department of ClinicalMicrobiology for their most valuable companionship, friendship and all their help during the years.Especially, my warmest thanks belong to Antti Ropponen, M.Sc., for his invaluable workcontribution and help during this study. I am also deeply grateful to Mikko Mättö, Ph.D., for hisguidance especially at the beginning of my research projects and help with flow cytometricmethods. My special thanks belong to Jonna Eeva, M.D., Ph.D., for all the fascinating discussions,critical review of the manuscripts and her support and friendship. I also wish to thank all the othermembers of our research group Ville Postila, M.D., Anna-Riikka Pietilä, M.Sc., Maija Seppä,M.D., Sanna Suoranta, Niina Simelius, M.Sc., Vi Lieu and Jemal Adem, M.Sc., for theircollaboration and help during this study. I am also deeply grateful to Pia Keinänen, RiittaKorhonen, Eila Pelkonen and Päivi Kivistö, for their excellent technical assistance and friendship.
I am deeply grateful to the official reviewers of this thesis, Docent Juha Klefström, Ph.D., andAdjunct Professor Kaisa Heiskanen, Ph.D., for their time, careful and critical evaluation of thethesis, most valuable comments and interesting research discussions on the phone.
I express my warm gratitude to all my dear friends for their support, discussions, laughs and cries,all the fun and enjoyable moments and adventures that we have experienced together.
I also express my warmest thanks to all my relatives. I owe my sincere thanks to my parents, Kerttuand Pekka, for all the help, never-ending love, enormous support and encouragement during mywhole life and just being there for me whenever I needed. I also wish to thank my grandfatherRudolf Böhme for all the support and love. I also want to express my sincere gratitude to my sisterEeva Kansala and her husband Kari Kansala for all the help, support and all the warm momentsthat we have shared, and my nieces Inka and Aada for bringing so much light, happiness and joyinto my life. My warm thanks also belong to my sister Tiina Nuutinen for her support and all thejoyful moments that we have shared.
This study was financially supported by the Kuopio University Hospital (EVO-fund), theUniversity of Kuopio, North-Savo Cancer Organization and Paavo Koistinen Foundation, whichare gratefully acknowledged.
Kuopio, December 2009
Ulla Nuutinen
ABBREVIATIONS
AIF apoptosis inducing factorALL acute lymphoblastic leukemiaApaf-1 apoptotic protease activating factor-1Bcl-2 B cell lymphoma 2 proteinBcl-XL Bcl-2 related gene (large variant)BCR B cell receptorBH BCL-2 homologyBIR baculoviral IAP repeatCARD caspase recruitment domainCaspase cysteinyl aspartate-specific proteinaseCHX cycloheximidec-IAP1 cellular IAP 1c-IAP2 cellular IAP 2CREB cyclic AMP-response element bindingcypD cyclophilin DCyt c cytochrome cDD death domainDED death effector domainDex dexamethasoneDIABLO direct IAP binding proteinDISC death inducing signaling complexDN dominant negativeDR death receptorGFP green fluorescent proteinEMSA electrophoretic mobility shift assayERK extracellular signal regulated kinaseFACS fluorescence activated cell sorterFADD Fas-associated death domainFDC follicular dendritic cellFL follicular lymphomaFLICE Fas-associated death-domain like interleukin-1 converting enzymeFLIP FLICE inhibitory proteinFOXO forkhead box OGC germinal center, glucocorticoidGR glucocorticoid receptorGRE glucocorticoid response elementGSK-3 glycogen synthase kinase-3HRP horseradish peroxidaseIAP inhibitor of apoptosis proteinIBM IAP-binding motifIg immunoglobulinI B inhibitor of BIKK I B kinaseILP-2 IAP-like protein 2IRES internal ribosomal entry siteJAK3 Janus family kinase 3
JNK JUN N-terminal kinaseIMM inner mitochondrial membraneLiCl lithium chloridemAb monoclonal antibodyMAPK mitogen activated protein kinaseMHC major histocombatibility complexML-IAP melanoma IAPMOMP mitochondrial outer membrane permeabilizationNAIP neuronal apoptosis inhibitory proteinNEMO NF- B essential modulatorNF- B nuclear factor BNIK NF- B inducing kinaseNLS nuclear localization sequenceNTD N-terminal domainOMM outer mitochondrial membraneOPG osteoprotegerinPBS phosphate buffered salinePDTC pyrrolidinedithiocarbamatePH pleckstrin homologyPI3K phosphatidylinositol 3-kinasePKB protein kinase BPLC phospholipase CPTEN phosphatase and tensin homologuePTP permeability transition poreRING really interesting new geneRIP receptor interacting proteinRT-PCR reverse transcriptase polymerase chain reactionSDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresisSLO secondary lymphoid organSmac second mitochondria-derived activator of caspaseSTAT3 signal transduced and activator of transcription 3tBid truncated BidTMRM methyl ester of tetramethylrhodamineTNF tumor necrosis factorTNFR tumor necrosis factor receptorTRADD TNF-receptor type 1-associated death domain proteinTRAF TNF receptor associated factorTRAIL TNF-related apoptosis inducing ligandt.u. transducing unitVDAC voltage dependent anion channelXIAP X-chromosome-linked IAP
m mitochondrial membrane potential
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, which will be referred to in the text by
Roman numerals.
I Nuutinen U, Postila V, Mättö M, Eeva J, Ropponen A, Eray M, Riikonen P, Pelkonen J.
(2006) Inhibition of PI3-kinase-Akt pathway enhances dexamethasone-induced apoptosis in
a human follicular lymphoma cell line. Exp Cell Res 312:322-330.
II Nuutinen U, Ropponen A, Suoranta S, Eeva J, Eray M, Pellinen R, Wahlfors J, Pelkonen J
(2009) Dexamethasone-induced apoptosis and up-regulation of Bim is dependent on
glycogen synthase kinase-3. Leuk Res. 33(12):1714-7.
III Nuutinen U, Simelius N, Ropponen A, Eeva J, Mättö M, Eray M, Pellinen R, Wahlfors J,
Pelkonen J (2009) PDTC enables type I signaling in type II follicular lymphoma cells. Leuk
Res. 33(6):829-36.
IV Nuutinen U, Ropponen A, Eeva J, Eray M, Pellinen R, Wahlfors J, Pelkonen J (2009) The
effect of microenvironmental CD40 signals on TRAIL- and drug-induced apoptosis in
follicular lymphoma cells. Scand J Immunol. In press.
The original publications (I-IV) have been reproduced with the permission of the publishers. In
addition, some unpublished data is presented.
CONTENTS
1. INTRODUCTION ..................................................................................................................... 17
2. REVIEW OF THE LITERATURE ......................................................................................... 18
2.1 B-cell development in periphery ........................................................................................... 182.1.1 Germinal centers ........................................................................................................... 182.1.2 CD40 signaling in germinal centers .............................................................................. 20
2.2 Follicular lymphoma ............................................................................................................. 212.2.1 General .......................................................................................................................... 212.2.2 Pathophysiology ............................................................................................................ 212.2.3 Treatment of FL ............................................................................................................ 22
2.3 Apoptosis ............................................................................................................................... 222.3.1 General .......................................................................................................................... 222.3.2 Caspases ........................................................................................................................ 23
2.3.2.1 Inhibitor of apoptosis proteins (IAPs) ................................................................... 232.3.3 Intrinsic apoptosis ......................................................................................................... 25
2.3.3.1 Mitochondrial outer membrane permeabilization .................................................. 252.3.3.2 Bcl-2 family proteins in the regulation of MOMP ................................................ 26
2.3.3.2.1 Members of the Bcl-2 family ......................................................................... 262.3.3.2.2 Activation of BH3-only proteins ................................................................... 272.3.3.2.3 Bax and Bak activation .................................................................................. 27
2.3.4 Extrinsic apoptosis ........................................................................................................ 292.3.4.1 TRAIL and its receptors ........................................................................................ 292.3.4.2 Physiological role of TRAIL ................................................................................. 302.3.4.3 TRAIL-induced apoptosis signaling ...................................................................... 302.3.4.4 TRAIL-induced non-apoptotic signaling ............................................................... 322.3.4.5 Mechanisms of TRAIL-sensitivity and resistance ................................................. 32
2.3.4.5.1 Decoy receptors ............................................................................................. 332.3.4.5.2 Post-translational regulation of TRAIL-receptors ......................................... 332.3.4.5.3 FLIPs ............................................................................................................. 332.3.4.5.4 Other mechanisms ......................................................................................... 34
2.3.4.6 Clinical trials of TRAIL ........................................................................................ 34
2.4 Glucocorticoid-induced apoptosis ........................................................................................ 342.4.1 General .......................................................................................................................... 342.4.2 Glucocorticoid receptor and function ............................................................................ 352.4.3 Regulation of transcription specific genes .................................................................... 362.4.4 Mechanisms of glucocorticoid-induced apoptosis ........................................................ 362.4.5 The role of lysosomes in GC-induced apoptosis ........................................................... 37
2.5 Regulation of apoptosis by PI3-kinase-Akt-pathway ............................................................. 372.5.1 General ........................................................................................................................... 372.5.2 Activation of PI3-kinase-Akt-pathway .......................................................................... 382.5.3 Anti-apoptotic targets of Akt ......................................................................................... 382.5.4 Glycogen synthase kinase-3 ........................................................................................... 40
2.5.4.1 General ................................................................................................................... 402.5.4.2 Regulation of glycogen synthase kinase-3 activity ................................................ 402.5.4.3 GSK3 promotes intrinsic apoptosis ........................................................................ 402.5.4.4 GSK3 inhibits extrinsic apoptosis .......................................................................... 41
3. AIMS OF THE STUDY ............................................................................................................. 42
4. MATERIALS AND METHODS .............................................................................................. 43
4.1 Cell lines and culture conditions (I-IV) ................................................................................. 43
4.2 Cell treatments (I-IV) ............................................................................................................. 43
4.3 3H-thymidine incorporation ................................................................................................... 44
4.4 Propidium iodide staining (I-IV) ........................................................................................... 44
4.5 Enzyme assay for caspase activity (I) .................................................................................... 44
4.6 Detection of changes in mitochondrial membrane potential ( m) ...................................... 454.6.1 ApoAlert Mitochondrial Membrane Sensor Kit (I)........................................................ 454.6.2 TMRM staining (II, III) ................................................................................................. 45
4.7 FACS analysis of receptors ................................................................................................... 45
4.8 Preparation of samples for Western blot analysis (I, III, IV) ................................................ 46
4.9 Preparation of mitochondrial and cytosolic fractions (I-III) ................................................. 46
4.10 Western blot analysis (I-IV) ................................................................................................. 464.10.1 Antibodies (I-IV) ......................................................................................................... 47
4.11 RT-PCR (IV) ........................................................................................................................ 47
4.12 Preparation of nuclear extracts (IV) ................................................................................... 48
4.13 EMSA assay (IV) .................................................................................................................. 48
4.14 Cloning (II-IV) ..................................................................................................................... 49
4.15 Site-directed mutagenesis, cloning and production of lentiviruses (II-IV) .......................... 49
4.16 Lentiviral transduction, FACS analysis and sorting of GFP-positive cells (II-IV) .............. 50
5. RESULTS AND DISCUSSION ................................................................................................ 51
5.1 Dexamethasone induces cell cycle arrest and apoptosis in dose-dependent manner(unpublished data) ...................................................................................................................... 51
5.2 Mechanism of Dex-induced apoptosis (I, II) ......................................................................... 53
5.3 Regulation of dexamethasone-induced apoptosis by PI3-kinase-Akt pathway (I) ................ 55
5.4 The role of glycogen synthase kinase-3 (GSK3) in Dex-induced apoptosis (II) .................... 58
5.5 Molecular mechanisms of TRAIL-induced apoptosis (III) .................................................... 595.5.1 Type I and type II TRAIL signaling (III) ...................................................................... 62
5.6 The effect of microenvironmental CD40 signaling on dexamethasone-, doxorubicin- andTRAIL-induced apoptosis (IV) .................................................................................................... 68
6. CONCLUSIONS ........................................................................................................................ 72
7. REFERENCES .......................................................................................................................... 75
APPENDIX: ORIGINAL PUBLICATIONS
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1. INTRODUCTION
Follicular lymphoma (FL) is one of the most common non-Hodgkin’s lymphoma worldwide. In
Finland, about 150 new cases are diagnosed annually. In the majority of FLs, the anti-apoptotic
mitochondrial Bcl-2 protein is overexpressed due to t(14;18) chromosomal translocation.
Microenvironment of FL B cells has also an important role in the progression of FL and might also
have an impact on the treatment. FL is a heterogeneous and presently still incurable malignancy,
characterized by a variable clinical course associated with frequent relapses and increasing
chemoresistance to conventional anti-cancer regimens. Therefore new approaches are urgently
needed for FL treatment.
The life and death of cells must be balanced to maintain tissue homeostasis. Cells have an intrinsic
mechanism to kill themselves via a process called apoptosis or programmed cell death. Apoptosis is
genetically regulated process that eliminates billions of unwanted or damaged cells every day in the
human body. Cells can undergo apoptosis via two different pathways: the intrinsic (mitochondrial)
or extrinsic (death receptor) pathway. Activity of either of the pathways eventually leads to the
activation of proteolytic cascade.
Apoptosis is also involved in the regulation of many pathological processes including cancer. One
of the hallmarks of human cancers is the evasion of apoptosis promoting tumor formation and
progression. The ability of tumor cells to evade engagement of apoptosis can also play a significant
role in their resistance to conventional therapeutic regimens.
Most approaches used in cancer therapy including chemotherapeutics kill cancer cells by inducing
apoptosis via the intrinsic pathway. In addition, tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) has emerged as the most promising for the death receptor targeted cancer therapy
due to its remarkable feature of selectively inducing apoptosis in cancer cells without causing
toxicity for normal cells. Recombinant TRAIL and monoclonal antibodies against TRAIL receptors
are now in phaseI/II clinical trials. Extensive research in apoptosis field continues to solve detailed
molecular mechanisms and regulation of apoptotic pathways, and is necessary to develop novel
therapeutic strategies either to kill cancer cells selectively or lower the apoptotic
threshold/overcome the resistance of cancer cells to molecules that induce apoptosis.
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2. REVIEW OF THE LITERATURE
2.1 B-cell development in periphery
The early B-cell development takes place in the bone marrow. B cells which have gained functional
non-autoreactive surface B cell receptor (BCR) pass selection process and leave the bone marrow.
These mature naive IgM and IgD expressing B cells circulate through the blood and lymph nodes
until they contact specific antigen in the T cell area in the secondary lymphoid organs (SLOs)
including spleen, lymph nodes, Peyer’s patches and tonsils. Antigens are brought to this location
from peripheral sites by macrophages and/or dendritic cells. After antigen contact B cells stop
migrating and remain in T cell zone (MacLennan, 1994).
B cells that recognize the antigens, internalize antigen via the BCR complex, process it to peptides
and present the peptide on MHC class II molecule to antigen specific helper T cells. Antigen
specific T cells form stable conjugates with the antigen presenting B cells and is followed by
soluble and membrane bound helper signals. These signals induce the B cells to proliferate and
most of them terminally differentiate into antibody-secreting plasma cells in specialized
extrafollicular sites (Klein and Dalla-Favera, 2008).
The minority of activated B cells migrates into the primary follicles and start dividing to form
germinal centers (Klein and Dalla-Favera, 2008). CD40 ligation (CD40 on B cells and CD40 ligand
(CD154) on T cells) induces germinal center pathway. The role of other molecular interactions and
cytokines remains controversial (MacLennan, 1994).
2.1.1 Germinal centers
Germinal centers are divided into mantle, dark and light zones. The mantle zone contains
recirculating B cells. Dark zone contains rapidly dividing centroblasts which undergo a massive
clonal expansion. During proliferation the somatic hypermutation is activated (MacLennan, 1994).
Somatic hypermutation introduces random point mutations into variable regions of the rearranged
heavy- and light-chain genes at very high rate, giving rise to mutant immunoglobulin molecules on
the surface of the B cells. Only some of the mutations increase the affinity of Ig for the antigen.
Hypermutation is necessery for the affinity maturation of antibody (Klein and Dalla-Favera, 2008).
Centroblasts continually give rise to the nondividing centrocytes of the light zone. Besides
centrocytes, the light zone also contains most of the follicular dendritic cells (FDC) and CD4+ T
cells that are present in germinal centers. Class switch recombination is thought to occur in light
19
zones of GCs. It ensures that the same variable region (the same antigen specificity) can be
expressed with different constant region isotypes. Isotype switching is directed by cytokines
released by helper T cells. CD40-CD154 interaction is also important for class switching
(Stavnezer, 2000).
Mature B cell
T helper cells
Antigen presentingdendritic cell
Plasma cellIgM/D
Germinal center
Mantle zone
centroblasts
Darkzone
Lightzone
Clonal expansionHypermutation
centrocytes
Class switchPositive and negativeselection
apoptosis
Plasma cell
Memory cell
FDC
Figure 1. B-cell development in periphery (see text for details).
In the light zone, centrocytes undergo selection process where new antigen receptors are tested.
Centrocytes which do not recognize the antigens anymore die by apoptosis. Centrocytes with best
affinity antigen receptors are selected by taking the antigen from follicular dendritic cells. Then
they process antigen and present it to T cells. T cells that recognize the antigens deliver two types
of stimuli: contact mediated stimuli and activating cytokines that induce the proliferation and
differentiation of B cells. B cells which have successfully bound antigen and survived the selection
leave the germinal center to either become memory B cells or high affinity antibody secreting
plasma cells (Klein and Dalla-Favera, 2008).
20
2.1.2 CD40 signaling in germinal centers
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is expressed on all
mature B cells including germinal center B cells. In vivo, T cells provide CD40 ligand for CD40
engagement on B cells (van Kooten and Banchereau, 2000). CD40L exists as a trimeric ligand and
the binding induces CD40 receptor oligomerization. This oligomerization appears to be a critical
initiating step for CD40 mediated signaling (Kehry, 1996).
CD40-mediated signaling is needed for B cell proliferation, isotype switching, germinal center
formation and memory B cell commitment in response to T cell dependent antigens. CD40
signaling also rescues the germinal center B cells as well as immature B cells from BCR-mediated
apoptosis (Dallman et al., 2003; Kehry, 1996). Mutations in the gene encoding CD40 ligand causes
an immunodefiency disease called X-linked hyper-IgM syndrome. It is characterized by normal or
elevated IgM levels but no IgG, IgA or IgE production, and defects in germinal center and memory
B-cell formation (DiSanto et al., 1993; Korthauer et al., 1993). A similar phenotype is seen in
CD40 or CD40L knockout mice (Dallman et al., 2003).
Upon ligand binding, the cytoplasmic tail of CD40 delivers signals to the cells by recruiting TNF
receptor-associated factors (TRAFs) 1, 2, 3, 5 and 6. The TRAF proteins activate different
signaling pathways including NF- B, mitogen activated protein kinases (MAPKs),
phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC ) pathway. In addition, the Janus
family kinase-3 (JAK3), which is associated with the cytoplasmic tail of CD40, undergoes
phosphorylation, resulting in the activation of the signal transducer and activator of transcription 3
(STAT3) (Elgueta et al., 2009).
The best-characterized signaling pathways activated by CD40 are the classical and the alternative
NF- B signaling pathways (Berberich et al., 1994; Coope et al., 2002; Homig-Holzel et al., 2008).
The ubiquitously expressed NF- B family of transcription factors includes NF- B1 (p105/p50),
NF- B2 (p100/p52), RelA (p65), RelB and c-Rel (Hayden and Ghosh, 2004). The activity of NF-
B is inhibited through the interaction with members of the I B family of proteins. Activation and
nuclear translocation of NF- B requires phosphorylation and proteosomal degradation of I Bs. In
classical pathway, I B is phosphorylated by the I B kinase (IKK) complex that contains two
catalytic subunits called IKK and IKK and a regulatory subunit called NEMO (NF- B essential
modulator). The degradation of I B complex induces the translocation of NF- B heterodimers
p50/p65 and p50/c-Rel to the nucleus. In contrast, the alternative NF- B pathway is activated
21
when NF- B inducing kinase (NIK) activates IKK , leading to processing of NF- B2/p100 to
generate p52, which associates with RelB and translocates to the nucleus (Elgueta et al., 2009).
Both signaling pathways regulate the transcription of several different genes by binding to
consensus B sites in promoters (Hayden and Ghosh, 2004). NF- B transcription factors may have
both anti-apoptotic and pro-apoptotic effects depending on the cell type and the type of the inducer.
The anti-apoptotic target genes of NF- B include inhibitors of apoptosis proteins (IAPs), FLICE-
inhibitory proteins (FLIPs) and Bcl-XL (Lee et al., 1999; Travert et al., 2008; Wang et al., 1998).
Although CD40 is considered a survival factor for normal B cells, in lymphoma B cells both CD40-
induced proliferation/survival and induction of growth arrest/apoptosis have been reported
(Dallman et al., 2003). CD40 ligation has been shown to decrease the proliferation or induce
apoptosis in high grade B cell lymphomas (Funakoshi et al., 1994; Wang et al., 2008). In contrast,
in low-grade B cell malignancies CD40 has been shown to promote survival (Ghia et al., 1998;
Travert et al., 2008).
2.2 Follicular lymphoma
2.2.1 General
Follicular lymphoma (FL) is the second most common non-Hodgkin’s lymphoma worldwide. It is
not curable and the median survival of FL patients is 8 to 10 years (de Jong, 2005). Most patients
are over 50 years of age at the time of diagnosis. FL is equally common in both genders. FL is
subdivided into three grades depending on the relative number of centroblasts in the lymphoma
samples. Grade 3 is further subdivided into 3a and 3b. Excluding the more aggressive Grade 3b
disease, FL is considered to be an indolent disease (Bendandi, 2008). Many patients are relatively
asymptomatic at the time of diagnosis and have usually only painless peripheral lymphadenopathy.
The patients with more advanced disease have symptoms such as fever, night sweats and weight
lose.
2.2.2 Pathophysiology
Majority of B cell lymphomas, including follicular lymphoma (FL), originate from GC B cells.
Most cases of follicular lymphoma have a t(14;18) translocation. The translocation juxtaposes the
bcl-2 gene on chromosome 18 and the enhancer region of the immunoglobulin gene from
chromosome 14. The enhancer region causes over-expression of bcl-2 gene. The Bcl-2 is an inner
22
mitochondrial membrane protein that blocks apoptosis. However, the Bcl-2 overexpression itself is
not sufficient to initiate tumorigenesis and other genomic alterations are needed for a malignant
transformation (de Jong, 2005). The fact that FL cells undergo spontaneous apoptosis when
cultured in vitro indicates that the microenvironment is also important for the development and
progression of FL in vivo (Alvaro et al., 2006; Dave et al., 2004; Glas et al., 2005). Like normal
germinal center B cells, FL cells interact through either soluble or membrane-bound signals with
various immune cells, such as follicular dendritic cells, T cells and macrophages within the tumor
microenvironment. CD40-CD40 ligand-interaction in combination with various cytokines has been
shown to prolong the in vitro survival of FL cells (Eray et al., 2003; Ghia et al., 1998). Gene
expression studies have shown that the nature of the tumor microenvironment predicts the survival
of patients with FL and may influence the response to treatments and risk of transformation (Dave
et al., 2004). The immune system may either promote or constrain tumor cell development,
depending on the relative distribution and activation status of various cell populations.
2.2.3 Treatment of FL
Clinical stage, prognostic factors and histological grading are needed to choose the best treatment.
Asymptomatic FL patients with slow-growing disease are usually not treated until symptoms
appear (wait-and-watch period). FL patients with a stage I or II disease are usually treated with
radiation. Patients with advanced-stage disease (stage III and IV) are treated with combination
chemotherapy or immunochemotherapy. In aggressive advanced stage FL chemotherapy is
extensively used. Initial treatment consist usually chlorambucil with or without prednisone. In
combination therapy, CVP (combination of cyclophosphamide, vincristine and prednisone) and
CHOP (combination of cyclophosphamide, doxorubicin, vincristine and prednisone) have been
widely used. A great improvenment in FL treatment has been achieved with the use of the chimeric
monoclonal anti-CD20 antibody (Rituximab). Autologous or allogeneic transplantation have been
used in young patients who relapse or show evidende of transformation (Bendandi, 2008).
2.3 Apoptosis
2.3.1 General
Programmed cell death or apoptosis is cell’s ability to kill itself in a controlled way. Apoptosis is
genetically regulated process and involved in regulation of many physiological and pathological
23
processes. During development, structures that are no longer needed are removed by apoptosis.
Throughout life, apoptosis eliminates cells that are useless or potentially dangerous such as aged,
infected, injured or mutated cells. Deregulation of apoptosis can lead to cancer (Jin and El-Deiry,
2005).
Apoptosis involves a series of biochemical events leading to typical morphological changes,
including cell shrinkage, membrane blebbing, cell membrane disruption, cytoskeletal
rearrangement, chromatin condensation, and DNA fragmentation. Apoptotic cells are usually
rapidly taken up and degraded by macrophages or neighboring cells before their intracellular
contents leak into the extracellular space, thereby avoiding an inflammatory response (Jin and El-
Deiry, 2005).
2.3.2 Caspases
Apoptosis is mediated by intracellular cysteine proteases called caspases which share the ability to
cleave their substrates after aspartate residues. To date, 14 mammalian caspases have been
identified of which caspases-2, -3, -6, -7, -8, -9, and -10 have been shown to be involved in
apoptosis (Li and Yuan, 2008). Caspases are produced as inactive zymogens containing a
prodomain, a p20 large subunit and a p10 small subunit. Most of the caspases are activated by
proteolytic cleavage. Active caspase is a heterotetramer, containing two small and two large
subunits (Li and Yuan, 2008).
Caspases are divided into initiator caspases (caspase-2, -8, -9, -10) and effector caspases (caspase-
3, -6, -7). Initiator caspases have long prodomains that contain the protein-protein interaction
motifs, DED (death effector domain) or CARD (caspase recruitment domain). DED and CARD are
involved in interacting with the upstream adaptor proteins. The short prodomain containing effector
caspases are typically cleaved and activated by upstream iniatiator caspases. Active effector
caspases are responsible for downstream execution steps of apoptosis by cleaving multiple cellular
substrates (Li and Yuan, 2008). To date over 400 caspase substrates are identified (Luthi and
Martin, 2007). There are two apoptosis signaling pathways, death receptor (extrinsic) and
mitochondrial (intrinsic) pathway of which activation leads to activation of caspases.
2.3.2.1 Inhibitor of apoptosis proteins (IAPs)
Enzymatic activity of caspases can be directly regulated by inhibitor of apoptosis proteins (IAPs).
The IAP family of proteins consists of eight human analogues, including cellular IAP1 (c-IAP1),
24
cellular IAP2 (c-IAP2), X-linked inhibitor of apoptosis (XIAP), survivin, Apollon (also known as
Bruce), melanoma IAP (ML-IAP, also known as Livin) and IAP-like protein 2 (ILP-2) (Hunter et
al., 2007).
Figure 2. Structure of human IAPs.
All IAP proteins contain one or more BIR (baculoviral IAP repeat) domains which are thought to
be responsible for caspase inhibition. In addition to the BIR domain, all IAPs except survivin
contain one or more other functional domains, for example, RING domain, which possesses E3
ubiquitin ligase activity, and the CARD domain (Nachmias et al., 2004).
XIAP is the best-described IAP and possibly the most potent suppressor of apoptosis. The linker
region that precedes the BIR2-domain binds to active site of caspase-3 or -7 and prevents substrate
binding. The BIR2 domain interacts with the N-terminus of the caspase small subunit. The BIR3
domain of XIAP binds to the IAP-binding motif (IBM) on the N-terminus of the small subunit of
caspase-9, which is exposed upon proteolytic processing of caspase-9. Distinct part of BIR3
domain heterodimerizes with an interface of caspase-9, which is required for homodimerization of
caspase-9. XIAP can also inhibit apoptosis through the E3 ubiquitin ligase activity of its RING
domain that mediates proteosomal degradation of proteins, including caspases and I B (Fulda,
2009).
In contrast to XIAP, the anti-apoptotic activity of c-IAP1 and c-IAP2 is thought not to be primarily
related to direct inhibition of caspases. Rather, they interfere with caspase activation by targeting
caspases or Smac/DIABLO to proteosomal degradation through their RING domain or binding to
Smac/DIABLO sequestering it from XIAP, thus facilitating XIAP-mediated inhibition of caspases.
25
In addition, c-IAP1 and c-IAP2 promote survival by inducing activation of NF- B (Samuel et al.,
2006; Fulda, 2009).
2.3.3 Intrinsic apoptosis
Intrinsic or mitochondrial apoptotic pathway is triggered by multiple stimuli including DNA
damage or cytotoxic drugs. The key event of mitochondrial pathway is mitochondrial outer
membrane permeabilization (MOMP) which results in the release of mitochondrial intermembrane
proteins such as cytochrome c (cyt c), second mitochondria-derived activator of caspase/ direct IAP
binding protein (Smac/DIABLO), HtrA2/Omi, apoptosis inducing factor (AIF) and endonuclease D
(EndoG) into cytosol. When released into the cytosol, cyt c binds to adaptor protein Apaf-1
(apoptotic protease activating factor-1). In the presence of dATP/ATP, the Apaf-1/cyt c complex
oligomerizes into a heptameric structure resembling a wheel whose core contains seven N-terminal
CARDs. The formation of this complex allows interaction of pro-caspase-9 with Apaf-1 through
the interaction of its own CARD with the CARD of Apaf-1, thus placing individual pro-caspase-9
molecules in close proximity with one another. In apoptosome caspase-9 becomes an active
initiator caspase and it further activates the effector caspases (caspase-3 and -7) which execute the
cell death program (Riedl and Salvesen, 2007; Li and Yuan, 2008).
Released Smac/DIABLO and serine protease HtrA2/Omi promote caspase activation by
counteracting IAP-mediated caspase inhibition. AIF and EndoG induce caspase-independent cell
death. AIF translocates to nucleus and causes chromatin condensation and high molecular weight
DNA fragmentation (Susin et al., 1999). EndoG is also translocated to nucleus and induce
internucleosomal DNA fragmentation (Li et al., 2001).
2.3.3.1 Mitochondrial outer membrane permeabilization
Mitochondria are cellular organelles involved in energy production and are essential for cellular
life. Mitochondria also play an important role in cell death. Mitochondrial outer membrane
permeabilization (MOMP) is considered to be the “point of no return” during apoptosis as it results
in the release of numerous apoptotic proteins that normally reside in the space between the outer
(OMM) and inner (IMM) mitochondrial membranes to the cytosol (Chipuk et al., 2006). MOMP is
frequently associated with the collapse of mitochondrial membrane potential ( m). Depending on
the stimuli, the loss of m can occur before, during or after MOMP (Green and Kroemer, 2004).
26
The mechanisms responsible for MOMP during apoptosis remain controversial. The most
important mechanism of MOMP under apoptotic conditions involves Bcl-2 family proteins. During
apoptosis, activated Bax and/or Bak form pores in the outer mitochondrial membrane (OMM)
resulting in the release of intermembrane proteins, such as cyt c, to the cytosol (activation of Bax
and Bak discussed below).
Inner mitochondrial membrane (IMM) can also participate in MOMP induction. Inner membrane
can cause MOMP through permeability transition pore (PTP) which is a complex composed of
several different proteins including VDAC (voltage dependent anion channel) in the OMM, ANT
(the adenine nucleotide translocator) in the IMM and cyclophilin D (cypD) in the matrix. It has
been suggested that opening of this pore leads to an influx of ions into the mitochondrial matrix
causing the loss of m, and swelling of the matrix as water enters leading to rupture of OMM and
MOMP (Chipuk et al., 2006; Green and Kroemer, 2004). However, PTP function in apoptosis is
still unclear because of swelling of mitochondrial matrix is not always a feature of apoptotic cells
and release of cyt c may precede or even occur in the absence of the loss of m (Skommer et al.,
2007). Instead, PTP formation appears to engage necrotic cell death (Green, 2005).
2.3.3.2 Bcl-2 family proteins in the regulation of MOMP
2.3.3.2.1 Members of the Bcl-2 family
The Bcl-2 family of proteins is divided into three groups based on their function and the number of
BCL-2 homology (BH) domains present. The anti-apoptotic members (Bcl-2, Bcl-XL, Bcl-w, Mcl-
1, A1 and Bcl-B) are associated with the outer mitochondrial membrane and protect cells against a
variety of apoptotic stimuli. They contain up to four BH domains (BH1-BH4). The pro-apoptotic
members are divided into two groups, Bax-like multidomain (BH1-BH3) apoptotic proteins (Bax,
Bak, Bok) and BH3-only proteins (Bik, Bid, Bad, Puma, Noxa, Bim, Hrk, Bmf). It is generally
believed that Bcl-2 family proteins form homo- and heterodimers and the interactions between pro-
and anti-apoptotic members neutralize the activity of each other (Adams and Cory, 2001; Bouillet
and Strasser, 2002; Korsmeyer, 1999; van Delft and Huang, 2006). Subsequently the ratio of pro-
apoptotic to anti-apoptotic Bcl-2 family members is critical in determining whether the cell will
undergo apoptosis.
27
2.3.3.2.2 Activation of BH3-only proteins
Activity of BH3-only proteins is regulated by several mechanisms at the transcriptional or at the
post-translational level. At least four BH3-only proteins are transcriptionally induced in response to
apoptotic stimuli. These include Hrk (Imaizumi et al., 1997; Imaizumi et al., 1999; Inohara et al.,
1997), Puma (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001), Noxa (Oda et al.,
2000) and Bim (Dijkers et al., 2000; Harris and Johnson, 2001; Putcha et al., 2001; Shinjyo et al.,
2001; Whitfield et al., 2001). Bad, Bim and Bik are regulated by phosphorylation (Zha et al., 1996;
Leung et al., 2008; Rambal et al., 2009; Verma et al., 2001). Pro-apoptotic potency of Bad and Bim
is decreased by phosphorylation. On the contrary, phosphorylation of Bik enhances its pro-
apoptotic activity (Verma et al., 2001). Bid is activated by proteolytic cleavage (Li et al., 1998; Luo
et al., 1998) and targeting of truncated Bid (tBid) to mitochondria is facilitated by N-myristoylation
at a site that becomes available for modification after cleavage (Zha et al., 2000). Bmf and Bim are
regulated by sequestration to cytoskeletal structures (Puthalakath et al., 1999; Puthalakath et al.,
2001). Bmf is sequestered to the actin cytoskeleton-based myosin V motor complex through its
interaction with dynein light chain DLC2 (Puthalakath et al., 2001). Bim has three isoforms of
which BimL and Bim EL are sequesterd to the microtubular dynein motor complex through
interaction with DLC1/LC8. BimS does not bind to DLC1 and this appears to be reason why it has
greater killing potency than BimL and BimEL (Puthalakath et al., 1999).
2.3.3.2.3 Bax and Bak activation
Bax and Bak are constitutively expressed and induce MOMP following apoptotic stimuli
suggesting that they are inactive in non-apoptotic cells (Kuwana and Newmeyer, 2003; Letai et al.,
2002). Bax proteins can be found as monomers in the cytosol or loosely associated with the outer
mitochondrial membrane when not activated. Bax translocates to and stably inserts into the
mitochondrial outer membrane during the activation process (Billen et al., 2008; Hsu et al., 1997;
Wolter et al., 1997). Bak is inserted into the outer mitochondrial membrane even when not
activated (Wei et al., 2000). Bax or Bak are necessary for mitochondrial apoptosis since cells from
mice lacking both Bax and Bak are resistant to a wide range of pro-apoptotic insults (Wei et al.,
2001; Zong et al., 2001). The BH3-only proteins are shown to be essential initiators of apoptosis
and they act upstream of Bax and Bak (Cheng et al., 2001; Huang and Strasser, 2000; Zong et al.,
2001). Activation of certain BH3-only proteins is required for Bax and Bak to oligomerize and
insert stably into the OMM.
28
There are two models how BH3-only proteins activate Bax and Bak. The direct activation model
suggests that certain BH3-only proteins, namely Bim, tBid and Puma, act as activators by binding
directly to Bax and Bak and promoting their activation (Cartron et al., 2004; Kim et al., 2006;
Kuwana et al., 2005; Letai et al., 2002; Marani et al., 2002; Oh et al., 2006; Walensky et al., 2006).
In this model, the rest of the BH3-only proteins act as sensitizers and bind to the anti-apoptotic Bcl-
2 members freeing Bim, tBid and Puma to activate Bax and Bak.
The indirect activation model suggests that all BH3-only proteins bind to anti-apoptotic Bcl-2
family proteins replacing Bax and Bak which leads to activation of Bax and Bak (Chen et al., 2005;
Willis et al., 2005; Willis et al., 2007). In this model, Bim, tBid and Puma are more potent
inducers of apoptosis since they can bind to all antiapoptotic Bcl-2 proteins (Certo et al., 2006;
Chen et al., 2005; Kuwana et al., 2005; Willis et al., 2005).
Figure 3. Direct and indirect activation model of how BH3-only proteins activate Bax/Bak.
29
Several recent findings strongly challenge the direct activation model (Willis et al., 2007).
Regardless which model of BH3-only function is correct, both lead to the activation of Bax and/or
Bak. One of the steps involved in the activation of Bax and Bak is a conformational change that
exposes the N-terminus of these proteins. Next step in this process is the formation of Bax and Bak
homo-oligomers that participate in forming pores and cause MOMP. However, the mechanisms by
which Bax and/or Bak form pores at the OMM and mediate MOMP are not fully understood.
2.3.4 Extrinsic apoptosis
The extrinsic apoptotic pathway is activated through engagement of the cell surface death receptors
by their ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) superfamily.
The members of this family are type I transmembrane proteins and are characterized by similar
cysteine-rich extracellular domains which defines their ligand binding. Death receptors contain an
intracellular 80-amino-acid-long region called “death domain” (DD) which is essential for
transduction of the apoptotic signal. The most studied death receptors are Fas (CD95/Apo-1),
TNFR1, TRAIL-R1 (DR4) and TRAIL-R2 (DR5/Killer/TRICK2). The ligands (FasL, TNF and
TRAIL) that bind to the death receptors are structurally related proteins that belong to the TNF
superfamily. These death ligands are mainly expressed as type II transmembrane proteins. In some
cases, these proteins can be proteolytically cleaved and released, although the apoptosis inducing
capacity of soluble ligands is significantly lower than of membrane-bound ligands (Guicciardi and
Gores, 2009).
2.3.4.1 TRAIL and its receptors
TRAIL was originally identified through sequence homology to the extracellular domain of FasL
and TNF (Pitti et al., 1996; Wiley et al., 1995). TRAIL forms homotrimers and interacts with three
cross-linked receptor molecules on the surface of the target cells. The biologically active
conformation of TRAIL is stabilized by a zinc ion positioned at the trimer interface (Hymowitz et
al., 2000). TRAIL induces apoptosis upon binding to its death domain containing receptors,
TRAIL-R1 or TRAIL-R2 (Chaudhary et al., 1997; Pan et al., 1997b; Walczak et al., 1997). TRAIL
can bind also to three other receptors which are unable to induce apoptosis and act as decoys.
Decoy receptors TRAIL-R3 (LIT, DcR1) and TRAIL-R4 (TRUNDD, DcR2) are expressed on the
surface of the cells but neither of them have a functional intracellular death domain (Marsters et al.,
1997; Pan et al., 1997a). The fifth TRAIL receptor is a soluble osteoprotegerin (OPG) (Emery et
30
al., 1998). However, TRAIL binding affinity for this receptor is low at physiological temperatures
(Truneh et al., 2000).
Figure 4. TRAIL receptors (see text for details).
2.3.4.2 Physiological role of TRAIL
The physiological function of TRAIL is largely unclear. TRAIL-R- and TRAIL-deficient knockout
mice are viable and fertile with no obvious developmental defects. TRAIL-R deficient mice have
enhanced innate immunity responses, indicating negative regulation of the innate immune system
via TRAIL (Diehl et al., 2004). Furthermore, TRAIL contributes to host immunosurveillance
against primary tumor development and metastasis (Cretney et al., 2002; Schmaltz et al., 2002;
Seki et al., 2003; Takeda et al., 2001). TRAIL knockout mice are more susceptible to tumor
initiation following carcinogen treatment and to tumor metastasis (Cretney et al., 2002) and
demonstrate an accelerated development of hematological malignancies (Zerafa et al., 2005).
TRAIL has also been implicated in the regulation of autoimmunity (Lub-de Hooge et al., 2005).
2.3.4.3 TRAIL-induced apoptosis signaling
Binding of trimeric TRAIL or agonistic monoclonal antibodies to TRAIL-R1 or TRAILR2 results
in receptor oligomerization on the cell membrane and initiation of apoptosis. The intracellular
signal transduction cascades triggered by FasL and TRAIL follow similar pathways. Activation of
31
the receptors leads to formation of a complex of proteins called death-inducing signaling complex
(DISC). DISC involves adaptor protein Fas associated death domain (FADD), which binds via its
own DD to the DD of the receptor and procaspase-8, which is recruited by FADD via interaction of
death effector domains (DEDs) that are present in both FADD and procaspase-8. At the DISC,
procaspase-8 is activated by auto-processing (Bodmer et al., 2000; Kischkel et al., 1995; Kischkel
et al., 2000; Sprick et al., 2000). In humans, caspase-10 can also be recruited to DISC and promote
apoptosis (Kischkel et al., 2001).
In Fas-induced apoptosis, the activation of caspase-8/10 may, depending on the cell type (type I or
II), lead either to direct activation of the downstream effector caspases or to amplification of the
signal by activation of the mitochondrial pathway (Scaffidi et al., 1998). In type I cells, strong
caspase-8/(10) activation at the DISC activates downstream effector caspases, such as caspase-3,-6
and -7, independently of mitochondria. In type II cells, caspase-8 activation is less efficient and
promotes cleavage of pro-apoptotic Bcl-2 family protein Bid. Truncated Bid (tBid) translocates to
mitochondria causing the depolarization of mitochondrial membrane and subsequent release of
mitochondrial apoptotic factors, such as cyt c to cytosol (Li et al., 1998; Scaffidi et al., 1998).
Subsequently, the apoptosome is formed leading to activation of caspase-9. Activated caspase-9
triggers downstream effector caspase activation, finally leading to apoptosis (Srinivasula et al.,
1998; Zou et al., 1999). It should be noted that in both type I and type II cells loss of mitochondrial
membrane potential and release of apoptotic factors from mitochondria are activated. However,
only in type II cells overexpression of Bcl-2 or Bcl-XL prevents apoptosis (Srinivasula et al., 1998;
Zou et al., 1999). Therefore, in type II cells apoptosis is mitochondria dependent and in type I cells
apoptosis can proceed independently of mitochondria.
TRAIL-induced apoptosis has also been shown to use both mitochondrial dependent and
independent pathways (Ozoren and El-Deiry, 2002; Suliman et al., 2001). Overexpression of Bcl-2
or Bcl-XL does not block TRAIL-induced apoptosis in some cancer cell lines (Fulda et al., 2002;
Keogh et al., 2000; Kim et al., 2001; Rudner et al., 2001; Walczak et al., 2000). In contrast,
overexpression of Bcl-2 or Bcl-XL blocks or reduces TRAIL-induced apoptosis in other cancer cell
lines (Fulda et al., 2002; Hinz et al., 2000; Munshi et al., 2001; Rokhlin et al., 2001; Ruiz de
Almodovar et al., 2001; Srinivasula et al., 2000; Sun et al., 2001).
32
Figure 5. Simplified model of type I and type II signaling pathways in TRAIL-induced apoptosis.
Interestingly, recently the lysosomal pathway and cathepsin B has also been connected to TRAIL-
induced apoptosis (Guicciardi et al., 2007; Nagaraj et al., 2006; Werneburg et al., 2007).
2.3.4.4 TRAIL-induced non-apoptotic signaling
In addition to triggering a pro-apoptotic signaling, TRAIL can activate multiple signal transduction
pathways such as MAPKs (extracellular signal regulated kinase (ERK), JUN N-terminal kinase
(JNK) and p38), PI3-kinase-Akt and NF- B. Various combinations of proteins are involved in
activation of these pathways by TRAIL. These proteins include FADD, TNF-receptor type 1-
associated death domain protein (TRADD), c-FLIP, caspases 8 and 10, TRAF2, NEMO and
receptor interacting protein (RIP) (Johnstone et al., 2008).
2.3.4.5 Mechanisms of TRAIL-sensitivity and resistance
TRAIL has been shown to induce apoptosis effectively in cancer cells with little or no toxicity
against normal cells. However, not all tumors are sensitive to TRAIL. Sensitivity of TRAIL-
induced apoptosis can be regulated at several levels.
33
2.3.4.5.1 Decoy receptors
Decoy receptors TRAIL-R3 and TRAIL-R4 can bind TRAIL but lack intracellular death domain
and therefore are incapable to mediate apoptosis. Initial observations that TRAIL-R3 and TRAIL-
R4 mRNA appeared to be primarily expressed on normal cells compared to tumour cells and that
overexpression of TRAIL-R3 or TRAIL-R4 inhibited TRAIL-induced apoptosis, indicated that the
relative expression of pro-apoptotic and decoy TRAIL-receptors could regulate the TRAIL
sensitivity (Marsters et al., 1997; Pan et al., 1997a; Sheridan et al., 1997). However, subsequent
studies using monoclonal antibodies specific for each receptor found no correlation between the
levels of TRAIL-R3 or TRAIL-R4 and sensitivity of cells to TRAIL (Griffith et al., 1999; Wagner
et al., 2007). It has been shown that TRAIL-R4 can suppress TRAIL-R2-mediated apoptosis by
forming a ligand independent (Clancy et al., 2005) or ligand-dependent (Merino et al., 2006)
complex.
2.3.4.5.2 Post-translational regulation of TRAIL-receptors
Novel resistance mechanism involving expression of peptidyl O-glycosyltransferase GALNT14
was recently identified (Wagner et al., 2007). Loss of this enzyme correlated with reduced
sensitivity to TRAIL because O-glycosylation of DR4 and DR5 promotes the ligand-stimulated
clustering of receptors leading to more efficient recruitment and activation of caspase-8 (Wagner et
al., 2007).
2.3.4.5.3 FLIPs
Activation of caspase-8 and -10 can be regulated by cellular FLICE-inhibitory protein (c-FLIP).
Several FLIP variants are generated by alternative splicing of the mRNA but only three of them are
expressed at the protein level, the most abundant c-FLIP long (c-FLIPL), a c-FLIP short (c-FLIPS)
and a short variant c-FLIPR. All of the isoforms have two DEDs and can be recruited to the DISC
through DED-DED interactions. c-FLIPL contains caspase-like domain but lacks catalytic cysteine
and therefore has no protease activity.
The role of c-FLIPS in inhibiting death receptor-mediated apoptosis is well established. c-FLIPS
was shown to block caspase-8 processing and activation at the DISC, probably competing for
binding to FADD. As c-FLIPR structure closely resembles c-FLIPS, it is likely that these proteins
inhibit death receptor-mediated apoptosis through similar mechanisms. On the contrary, the
34
function of c-FLIPL at the DISC remains controversial. Originally, c-FLIPL was described as an
anti-apoptotic protein that inhibits death receptor induced apoptosis by interfering with caspase-8
activation at the DISC. Overexpression of c-FLIPL results in the recruitment of both c-FLIPL and
procaspase-8 to the DISC, followed by defective processing of caspase-8. In addition, several
studies have implicated c-FLIPL in the activation of the survival signaling pathways, especially NF-
B-pathway. On the contrary, some studies describe pro-apoptotic functions of c-FLIPL, referring
to its assistance in the auto-catalytic activation of pro-caspase-8 at the DISC (Micheau et al., 2002).
2.3.4.5.4 Other mechanisms
TRAIL-resistance can also result from reduced caspase-8 expression (Eramo et al., 2005; Hopkins-
Donaldson et al., 2000), increase expression of IAPs such as XIAP (Cummins et al., 2004) and c-
IAP2 (Ricci et al., 2007) or overexpression of anti-apoptotic Bcl-2 members including Bcl-2, Bcl-
XL and Mcl-1 (Fulda et al., 2002; Hinz et al., 2000; Ricci et al., 2007; Rosato et al., 2007).
2.3.4.6 Clinical trials of TRAIL
Preclinical studies have established that using nontagged, soluble zinc-replete form of TRAIL is
effective, nontoxic on various organs, including the liver, and, therefore suitable for clinical
studies. As a result, soluble recombinant TRAIL (rh-APO2/TRAIL) and agonistic antibodies
against TRAIL-R1 (mabatumumab or HGS-ERT1) or TRAIL-R2 receptors (lexatumumab or HGS-
ERT2, AMG 655, apomab) are currently in PhaseI/II clinical trials for treatment of solid tumors
and hematological malignancies (Johnstone et al., 2008).
2.4 Glucocorticoid-induced apoptosis
2.4.1 General
Glucocorticoids (GCs) are a class of steroid hormones that exert a wide range of anti-inflammatory
and immune-suppressive activities. Therefore, GCs such as dexamethasone are commonly used in
the treatment of inflammatory and autoimmune diseases. In addition, the ability of GCs to induce
cell cycle arrest and apoptosis in lymphoid cells, has led to their inclusion in chemotherapy
protocols for lymphoid malignancies (Alexanian and Dimopoulos, 1994; Gaynon and Carrel, 1999;
Moalli and Rosen, 1994).
35
2.4.2 Glucocorticoid receptor and function
The effects of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the
steroid hormone receptor superfamily that functions as ligand dependent transcription factor. GR
consists of three distinct structural and functional domains. N-terminal region domain (NTD)
contains a ligand independent transactivation domain. A central DNA binding domain consists of
two highly conserved zinc finger domains and is essential for binding to glucocorticoid response
element (GRE) sequences of regulated genes. This domain contains also nuclear localization
sequence (NLS). The first zinc finger domain is necessary for binding to NF- B and AP-1 and for
the transrepression function of the GR. The second zinc finger domain is involved in receptor
dimerization and GRE transactivation. The C-terminal region contains the ligand binding domain
which is also required for binding heat-shock proteins and GR dimerization. It also contains a
ligand-dependent transactivation domain and nuclear localization signal (Frankfurt and Rosen,
2004).
In its unactivated state, GR associates with heat shock proteins in the cytoplasm. Upon ligand
binding, GR dissociates from the complex and translocates to the nucleus, and either increases or
decreases gene expression (Herr et al., 2007).
Figure 6. Activation of GR.
Gene induction of GR is mediated via GR interaction with conserved GREs, whereas gene
repression occurs through negative GREs, protein-protein interaction with other transcription
factors, competition for co-activators and other mechanisms. GCs can also exert more immediate,
36
presumably non-genomic but still GR-dependent, effects. However these mechanisms are less well
understood. In addition to the well-characterized cytoplasmic/nuclear GR, a membrane-associated
GR was reported but its existence and possible significance remained controversial (Schmidt et al.,
2004).
GC-induced apoptosis is strictly dependent on the interaction of GC with its receptor. The
requirement for the GR has been shown in thymocytes from genetically modified mice, and human
acute lymphoblastic leukemia (ALL) cell lines with mutated GR and by conferring GC sensitivity
to GC-resistant ALL-cell lines by GR transgenesis (Schmidt et al., 2004). Additionally, the level of
GR expression is a critical determinant for GC sensitivity and GR autoinduction is required for GC-
induced apoptosis (Kofler et al., 2003; Pedersen and Vedeckis, 2003; Ramdas et al., 1999; Riml et
al., 2004).
2.4.3 Regulation of transcription specific genes
It is widely accepted that GC-induced apoptosis results from alterations in gene expression.
However, it is still controversial whether it requires gene transactivation, transrepression or both
(Schmidt et al., 2004). Several studies have identified a large number (over 900) of genes during
GC-induced apoptosis by using expression profiling and various model systems of GC-induced cell
death (Schmidt et al., 2004). However distinct set of genes were regulated in different cell systems
and experimental conditions indicating that multiple cell-context-dependent mechanisms rather
than a conserved pathway may lead to GC-induced cell death. Some of these genes are directly
implicated in death and survival decisions (such as Bim and granzyme A) or in cellular stress (such
as I B and c-myc). However, some of the genes are not causal in the death response.
Upregulation of GR itself was also shown (Schmidt et al., 2004).
2.4.4 Mechanisms of glucocorticoid-induced apoptosis
GC has been shown to activate the apoptotic machinery by regulating components of either the
extrinsic or intrinsic pathways or both. It has been shown that inhibition of caspase-8 prevented
GC-induced release of cyt c and apoptosis (Marchetti et al., 2003). However, the involvement of
death receptor pathway has been questioned and it has been suggested that caspase-8 is activated
downstream from mitochondria since activation of caspase-8 was markedly reduced in Apaf-
deficient thymocytes treated with dexamethasone while death receptor-mediated activation of
37
caspase-8 was not diminished (Herr et al., 2007). Additionally, GC-induced apoptosis was
unaffected in Bid-deficient mice (Yin et al., 1999).
The involvelvement of the intrinsic pathway in GC-induced apoptosis is evident. In thymocytes
from caspase-9 deficient mice GC-induced apoptosis and also the loss of mitochondrial membrane
potential were prevented (Hakem et al., 1998; Kuida et al., 1998). In addition, it has been shown
that dexamethasone induces loss of mitochondrial membrane potential in thymocytes and T cell
hybridoma cells (Camilleri-Broet et al., 1998; Marchetti et al., 1996; Petit et al., 1995; Yoshino et
al., 2001). Furthermore, overexpression of antiapoptotic Bcl-2 proteins attenuated GC-induced cell
death in mouse thymocytes, human ALL and multiple myeloma cell lines (Feinman et al., 1999;
Grillot et al., 1995; Hartmann et al., 1999). Since induction of proapoptotic proteins Bim (Abrams
et al., 2004; Bachmann et al., 2005; Planey et al., 2003; Wang et al., 2003; Zhang and Insel, 2004)
and Puma (Erlacher et al., 2005) as well as repression of antiapoptotic Bcl-2 and Bcl-XL (Casale et
al., 2003; Chauhan et al., 2002) proteins have been observed in GC-treated cells, transcriptional
regulation of Bcl-2 proteins may be essential for GC-induced apoptosis in many systems.
2.4.5 The role of lysosomes in GC-induced apoptosis
Lysosomes may play an important role in both caspase-dependent and caspase-independent cell
death (Kroemer and Jaattela, 2005). Central mediators in this process are cathepsins (such as
cathepsin B), which are synthesized as proenzymes, transported into the lysosomal vesicle, and
activated through proteolytic cleavage. As a result of lysosomal membrane permeabilization
(LMP), cathepsin B is released from lysosomes to the cytosol, where it can induce cell death by
activating initiator caspases or directly cleaving nuclear substrates (Kroemer and Jaattela, 2005).
Cathepsin B activation was shown to be an early and essential step in the execution phase of GC-
induced apoptosis in thymocytes. It was postulated that GC-induced activation of caspase-3 by
caspase-9 in thymocytes occurs directly and also indirectly through a lysosomal amplification loop
(Wang et al., 2006).
2.5 Regulation of apoptosis by PI3-kinase-Akt-pathway
2.5.1 General
The phosphatidyl-inositol-3-kinase (PI3K)-Akt signaling pathway regulates fundamental cellular
functions such as transcription, translation, proliferation, growth and survival. Disturbed activation
38
of PI3K-Akt pathway has been associated with development of cancer. Indeed, alterations in the
PI3-kinase-Akt signaling pathway are frequent in human cancers. It has been suggested that
constitutively active Akt promotes cellular survival and resistance to chemotherapy in solid tumors
(Brognard et al., 2001; Clark et al., 2002; Ng et al., 2000; West et al., 2002) and in cells of
hematopoietic lineage (Cataldi et al., 2001; Martelli et al., 2003; Mitsiades et al., 2002; O'Gorman
et al., 2000).
2.5.2 Activation of PI3-kinase-Akt-pathway
PI3K is a phospholipid-modifying enzyme that phosphorylates the phosphoinositols at the 3´
position of the inositol ring. It exists as a heterodimer of catalytic subunit (p110) and regulatory
subunit (p85). Activation of the PI3-kinase signaling pathway involves recruiting PI3-kinase to the
plasma membrane, where its substrates are located and tyrosine phosphorylation of PI3-kinase.
Activation of PI3K leads to accumulation of two PI3-kinase products, PI(3,4)-P2 and PIP3. PI(3,4)-
P2 and PIP3 act directly as second messengers and can bind to and activate several protein kinases.
PIP3 can also recruit cytoplasmic signaling proteins to the plasma membrane by binding to their
pleckstrin homology (PH) domains (Franke, 2008).
The downstream target of PI3-kinase pathway is serine/threonine kinase Akt, also called protein
kinase B (PKB). Binding of PIP3 to the PH domain of Akt is important for recruiting Akt to the
plasma membrane where it can be phosphorylated at two residues (Thr308 and Ser473) by
upstream kinases and thereby activated (Franke, 2008).
Negative regulation of the PI3-kinase-Akt pathway is mainly accomplished by phosphatase and
tensin homologue (PTEN). PTEN is a dual-specificity phosphatase that has activity against lipid
and protein substrates. The main physiological target of PTEN is PIP3, which is one of the PI3K
products. PTEN reduces the amount of PIP3 and changes it to PIP2 through dephosphorylation of 3´
inositol position. Inactivating mutations or a loss of PTEN expression leads to increased activation
of Akt followed by increased cell proliferation and resistance to apoptosis (Osaki et al., 2004).
2.5.3 Anti-apoptotic targets of Akt
Activated Akt has several important functions including prevention of apoptosis. One way in which
Akt may prevent apoptosis is by phosphorylating BH3-only protein Bad (at Ser-136).
Phosphorylation of Bad creates a binding site for a member of the 14-3-3 protein family. When Bad
is bound to 14-3-3, it cannot promote apoptosis (Zha et al., 1996).
39
Phosphorylation of forkhead family of transcription factors by Akt alters their intracellular
localization. In the absence of Akt activation, FOXO proteins are predominantly localized in the
nucleus where they are able to promote transcription of proapoptotic targets genes such Fas ligand,
Bim and Puma (Brunet et al., 1999; Dijkers et al., 2000; You et al., 2006). When phosphorylated by
Akt, FOXOs are exported from the nucleus and sequesterd by 14-3-3 proteins in the cytosol
(Brunet et al., 1999).
Akt has also been shown to directly phosphorylate procaspase-9 on Ser-196, and this
phosphorylation correlates with the decrease in the protease activation of caspase-9 (Cardone et al.,
1998). X-linked inhibitor of apoptosis protein (XIAP) has been suggested as potential Akt target
since it has been shown that Akt phosphorylates and stabilizes XIAP (Dan et al., 2004).
Akt can phosphorylate and activate I B kinase (IKK ), which in turn phosphorylates I B,
targeting it for degradation (Kane et al., 1999; Ozes et al., 1999). This leads to nuclear translocation
and activation of nuclear factor- B (NF- B). In addition, Akt phosphorylates and activates the
cyclic AMP-response element-binding (CREB) protein, which increases the transcription of anti-
apoptotic genes, such as Bcl-2 and Mcl-1 (Du and Montminy, 1998; Pugazhenthi et al., 2000;
Wang et al., 1999).
Interestingly, Akt regulates indirectly tumor suppressor p53 protein, which acts as a cellular sensor
of cellular stress and transduces stress signals into apoptotic signals. Akt promotes phosphorylation
and nuclear translocation of Mdm2 (Hdm2 in humans) which destabilizes p53 leading to
degradation of p53 (Mayo and Donner, 2001; Zhou et al., 2001).
Figure 7. Anti-apoptotic targets of Akt.
40
2.5.4 Glycogen synthase kinase-3
2.5.4.1 General
Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase which is ubiquitously expressed in
mammalian cells. Two GSK3 isoforms encoded by distinct genes have been identified, GSK3 and
GSK3 . Initially, GSK3 was identified as a kinase that phosphorylates and inactivates a key
metabolic enzyme, glycogen synthase. Subsequently GSK3 has been shown to be involved also in
several other cellular processes, including apoptosis. Interestingly, GSK3 has the capacity to either
increase or decrease the threshold for apoptosis (Beurel and Jope, 2006).
2.5.4.2 Regulation of glycogen synthase kinase-3 activity
The activity of GSK3 is tightly controlled by multiple mechanisms. Phosphorylation of Ser-21 in
GSK3- or Ser-9 in GSK3- inhibits its activity. PI3-kinase-Akt signaling pathway is a major
regulator of GSK3 because Akt phosphorylates GSK3 on these serine residues. However, several
other kinases have also been shown to phosphorylate them. Conversely, the enzymatic activity is
enhanced by phosphorylation of tyrosine-279 in GSK3- or tyrosine-216 in GSK3- . However, the
mechanisms regulating these phosphorylations are not well-defined. In addition to phosphorylation,
GSK3 activity can be regulated by complex formation and by the control of its intracellular
localization. Additionally, the action of GSK-3 is usually indirectly controlled by the
phosphorylation state of its substrate since the most substrates of GSK-3 must be pre-
phosphorylated (Beurel and Jope, 2006).
2.5.4.3 GSK3 promotes intrinsic apoptosis
GSK3 has been shown to promote mitochondrial intrinsic apoptosis. Overexpression of GSK3 was
found to induce apoptosis (Pap and Cooper, 1998). After that, activation of GSK-3 has been shown
to promote apoptosis induced by various stimuli, including staurosporine (Bijur et al., 2000),
inhibition of PI3-kinase (Pap and Cooper, 1998), serum deprivation (Linseman et al., 2004),
mitochondrial toxins (King et al., 2001) and DNA damage (Beurel et al., 2004; Beurel et al., 2005;
Jin et al., 2005; Tan et al., 2006; Watcharasit et al., 2002; Watcharasit et al., 2003). It has been
shown that GSK3 is present in mitochondria and mitochondrial GSK3 activity is increased during
intrinsic apoptotic signaling induced by DNA damage or ER stress (Bijur and Jope, 2003). Direct
41
mitochondrial substrates have not yet been found. However, studies have shown that its apoptotic
action is upstream of caspase-9 (Bijur et al., 2000), cytochrome c release from mitochondria
(Watcharasit et al., 2003) and PTP complex in mitochondria (Juhaszova et al., 2004). In addition,
GSK3 can directly phosphorylate Bax, which results in activation of Bax (Linseman et al., 2004).
Phosphorylation by GSK3 enhances degradation of Mcl-1 (Maurer et al., 2006). GSK3 also
phosphorylates VDAC which prevents hexokinase II from associating with VDAC which may
faciliate the mitochondrial association of pro-apoptotic Bcl-2 family members to promote apoptosis
(Cheng et al., 2003; Majewski et al., 2004; Pastorino et al., 2002). Mitochondrial function during
intrinsic apoptosis signaling is also influenced by GSK3-mediated regulation of the expression of
proteins participating in the mitochondrial stage of apoptosis. GSK3 regulates in the nucleus
transcription factors p53 and CREB. GSK3 activity promotes p53-induced expression of Bax in
response to DNA damage (Tan et al., 2005; Watcharasit et al., 2003) and inhibits CREB-dependent
expression of Bcl-2 (Bullock and Habener, 1998; Grimes and Jope, 2001). GSK3 has also been
shown to be required for stress-induced expression of Bim (Hongisto et al., 2003).
2.5.4.4 GSK3 inhibits extrinsic apoptosis
GSK3 has an opposite effect on extrinsic apoptosis inhibiting the death-receptor-mediated
apoptosis. It has been shown in human prostate cancer cell lines that lithium and another highly
selective inhibitor of GSK3, SB216763, as well as by knockdown of the GSK3 protein level using
RNA interference potentiates TRAIL-induced apoptosis (Liao et al., 2003). Apoptosis induced by
stimulation of DR5 with an agonistic antibody was shown to be potentiated by inhibition of GSK3
(Rottmann et al., 2005). In addition, apoptosis induced by Fas was found to be regulated by the
anti-apoptotic action of GSK3 since several inhibitors of GSK3 potentiated Fas-induced apoptosis
in Jurkat cells and in neurons (Song et al., 2004). Several studies have shown that inhibition of
GSK3 enhances the death receptor apoptosis already upstream of the activation of caspase-8 (Aza-
Blanc et al., 2003; Liao et al., 2003; Schwabe and Brenner, 2002). The precise mechanism remains
to be identified.
42
3. AIMS OF THE STUDY
The overall aim of this study was to investigate detailed molecular mechanisms and regulation of
mitochondrial and death receptor apoptotic pathways in human FL cell lines. Detailed knowledge
of the regulatory mechanisms of the apoptotic pathways is necessary for development of novel
therapeutic strategies for FL. In addition, as germinal center microenvironment of FL cells,
especially CD40 signaling, could have an impact on the treatment, the effect of CD40 signaling on
TRAIL- and drug-induced apoptosis was evaluated.
The specific aims were
1. To examine the detailed molecular mechanisms of dexamethasone (Dex)-induced
apoptosis and the involvement of known survival pathways in the regulation of Dex-
induced apoptosis in human follicular lymphoma cell line (I).
2. To elucidate the requirement of mitochondria and the role of glycogen synthase kinase-3
(GSK3) in dexamethasone-induced apoptosis (II).
3. To determine type I and type II signaling pathways in TRAIL-induced apoptosis (III).
4. To investigate whether the CD40-CD40L interactions between FL cells and the cells in
their GC microenvironment could have an impact on the treatment of FL and to elucidate
how the possible anti-apoptotic function of CD40 could be counteracted (IV).
43
4. MATERIALS AND METHODS
4.1 Cell lines and culture conditions (I-IV)
All the experiments have been performed on human follicular lymphoma cell lines HF1A3,
HF28RA and HF4.9 (Eray et al., 2003; Matto et al., 2005). The cell lines have been established
from enlarged lymph nodes of FL patients. The origin and characteristics of the cell lines have
been described previously (Eray et al., 2003). As a hallmark of a FL, all three cell lines carry the
t(14;18) translocation and overexpress Bcl-2 protein (Eray et al., 2003). Cells were cultured at
37 C in a moist atmosphere of 5 % CO2 in RPMI 1640 media (Gibco BRL Life Sciences,
Paisley, Scotland) supplemented with 5 % heat-inactivated fetal calf serum (FCS, Biological
Industries, Kibbuz Beit Haemek, Israel), 2 mM L-glutamine, 200 µg/ml streptomycin (Sigma,
Stenheim, Germany), 240 IU/ml penicillin (Orion, Espoo, Finland), 10 mM HEPES buffer
(Gibco BRL), 0.1 mM nonessential amino acids (Gibco BRL), 1.0 mM sodium pyruvate (Gibco
BRL), and 20 µM 2-mercaptoethanol (Fluka Chemie, Buch, Switzerland).
4.2 Cell treatments (I-IV)
Dexamethasone was from Sigma-Aldrich (Stenheim, Germany) (I, II, IV). Inhibitors of signaling
pathways PI3-kinase inhibitor LY294002, Akt-inhibitor (1L-6-hydroxy-methyl-chiro-inositol 2(R)-
2-O-methyl-3-O-octadecylcarbonate), MEK inhibitor PD98059, protein kinase C (PKC) inhibitor
Gö6850 and Raf Kinase Inhibitor ZM336372, were purchased from Calbiochem (La Jolla, CA) (I).
Protein synthesis inhibitor cycloheximide (CHX) was purchased from Sigma-Aldrich (I). SB-
216763 (Sigma-Aldrich) and LiCl (Sigma-Aldrich) were used to inhibit GSK3 (II). His-tagged
recombinant human soluble Killer TRAIL was obtained from Apotech (Epalinges, Switzerland)
(III, IV). Doxorubicin hydrochloride solution was obtained from Sigma-Aldrich (IV).
Pyrrolidinedithiocarbamate (PDTC) (Sigma-Aldrich) and IKK inhibitor III (BMS-345541)
(Calbiochem, La Jolla, CA) were used to inhibit NF- B pathway (II, IV). The anti-CD40 mouse
mAb AF1.15 was gently provided by Dr Matti Kaartinen from Haartman institute, University of
Helsinki, Finland (IV). Anti-TRAIL-R1 (clone HS101) and anti-TRAIL-R2 (clone HS201)
antibodies (Abs) (Alexis Biochemicals, Gruenberg, Germany) were used for FACS staining and
receptor blockade.
44
4.3 3H-thymidine incorporation
Thymidine incorporation was carried out using 6 x 10E4 cells/well in 200 µl of medium containing
appropriate stimuli. After 20 h of incubation, 3µCi of (3H) thymidine (specific activity 2.0
Ci/mmol, Amersham Biosciences, Piscataway, NJ) was added to each well for 4 h. Cells were
harvested and the incorporated radioactivity was detected by beta scintillation counting.
4.4 Propidium iodide staining (I-IV)
DNA fragmentation was detected by flow cytometric analysis after propidium iodide staining. Cells
with fragmented DNA were considered as apoptotic. Fixation and staining of the cells were
performed according to standard protocols. Briefly, samples containing 1 x 106 cells were
collected, resuspended in ice-cold PBS and fixed with ethanol (70 % v/v). After overnight
incubation at +4 C, cells were centrifuged and incubated with 150 g/ml RNase for 1 h at 50 C.
Propidium iodide (PI) (Molecular Probes, Leiden, Netherlands) was added to the final
concentration of 8 g/ml and incubation was continued for 2 h at +37 C. FACScan flow cytometer
with CellQuest v 3.1 software (Becton Dickinson, Mountain View, CA) was used in the analysis.
4.5 Enzyme assay for caspase activity (I)
Activity of caspase-3 was analyzed by measuring the cleavage rate of a synthetic fluorescent
substrate Ac-DEVD-AMC (Calbiochem). The protease assay was carried out according to the
manufacturer’s instructions. Briefly, 2 x 106 cells per sample were lyzed in 100 l of lysis buffer
(50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 10mM NaH2HPO4, 10mM Na2HPO4, 1%
Nonidet P-40, 0.1 mM PMSF, 1 M VO4, 5 g/ml aprotinin, 5 g/ml leupeptin). After 30 min of
incubation on ice, the cell lysate was centrifuged (10,000 g, 30 min) and the supernatant was used
as a cytosolic extract. The concentration of cytosolic proteins was measured by Lowry’s method
(DC protein assay, Bio-Rad laboratories, Hercules, CA). Equal amount of protein was dissolved in
protease assay buffer (20 mM Hepes, 10 % glycerol, 2mM DTT). Samples were incubated with 20
M Ac-DEVD-AMC fluorogenic substrate for 2 h. After incubation, released fluorogenic AMC
was measured using Wallac Victor 1420 spectrofluorometer (Wallac, Turku, Finland) with an
excitation wavelength of 355 nm and emission wavelength of 460 nm.
45
4.6 Detection of changes in mitochondrial membrane potential ( m)
4.6.1 ApoAlert Mitochondrial Membrane Sensor Kit (I)
ApoAlert Mitochondrial Membrane Sensor Kit (Clontech Laboratories, Inc., Palo Alto, CA) was
used for detecting changes in m during apoptosis. The kit contains a cationic dye
MitoSensorTM that fluoresces differently in apoptotic and non-apoptotic cells, because of changes
in mitochondrial membrane permeability. Cells were stained and analyzed according to
manufacturer’s instructions. Briefly, 5 x 105 cells were collected and resuspended in 500 l of the
incubation buffer containing 0.5 l MitoSensor TM reagent and incubated for 15 min at 37 C in a
5 % CO2 incubator. After staining cells were washed with and resuspended in the Hepes buffer
(10 mM Hepes, 140 mM NaCl, 5 mM CaCl2, pH 7.4) and the fluorescence signal was analyzed
by flow cytometry. Forward and side scatter was used for the selection of early apoptotic cells.
4.6.2 TMRM staining (II, III)
Depolarization of mitochondrial membrane was detected by TMRM staining. TMRM is the methyl
ester of tetramethylrhodamine and it is detected in FL2 channel. After incubation of appropriate
stimuli, cells (1x10E6) were stained with 150 nM TMRM (Molecular Probes) for 15 min at 37 °C
in the dark. After staining cells were immediately analyzed with FACScan flow cytometer (Becton
Dickinson). Forward and side scatter were used to gate living/early apoptotic cells for TMRM
analysis.
4.7 FACS analysis of receptors
Cells (5x105) were incubated with mAbs against TRAILR1 and TRAILR2 at the concentration of
1µg in 100 µl of FACS-buffer (2% FCS in PBS) followed by PE-labeled goat anti-mouse (BD
Pharmingen) at the concentration of 0.4 µg/ml. Stained cells were analyzed using FACScan flow
cytometry equipped with a CellQuest data analysis program (Becton Dickinson, San Diego, CA,
USA). Isotype matched (mouse IgG1) antibody was used to control the unspecific binding of
antibodies.
46
4.8 Preparation of samples for Western blot analysis (I, III, IV)
After stimulation, cells (2 106) were collected and resuspended with lysis buffer (20 mM
Tris/HCl (pH 8.0), 2 mM EDTA, 3% NP-40, 100 mM NaCl, 50 mM NaF, 1 M PMSF, 1 M VO4,
5 g/ml aprotinin, 5 g/ml leupeptin). After 30 min incubation on ice samples were centifuged at
10 000 g, 15 min, + 4 C. Protein concentration of the samples was measured and equalized with
SDS-PAGE buffer (0.125 M Tris-Cl pH 6.8, 4 % SDS, 20 % glyserol, 10 % 2-mercaptoethanol,
bromofenolblue).
4.9 Preparation of mitochondrial and cytosolic fractions (I-III)
Mitochondrial and cytosolic fractions were obtained using ApoAlertTM cell fractionation kit
(Clontech Laboratories) according to the manufacturer’s protocol. In brief, 2 107 cells were
stimulated harvested and washed with wash buffer. The cell pellet was resuspended in 0.8 ml
fractionation buffer containing protease inhibitors and DTT, and incubated on ice for 10 min.
Subsequently, cells were homogenized by passing the cell suspension through a 27G syringe
needle. The homogenate was centrifuged at 700 g for 10 min at 4 C. The remaining supernatant
was centrifuged at 10,000 g for 25 min at 4 C. The supernatant (a cytosolic fraction) was
collected and the pellet (mitochondrial fraction) was resuspended in 0.1 ml of the fractionation
buffer. The protein concentration of fractions was determined and equalized with SDS-PAGE
buffer.
4.10 Western blot analysis (I-IV)
Equal amount of protein from each sample was separated on 12 %-15% SDS-PAGE gel and
transferred to nitrocellulose membranes (Hybond ECL nitrocellulose membranes, Amersham
Biosciences). Membranes were blocked overnight or for 1h in PBS containing 3% BSA and 0.1%
Tween or 5 % milk and 0.1% Tween at room temperature and incubated for 2 h with primary
antibodies. After washing, the membranes were incubated for 30 min-1 h with horseradish
peroxidase-conjugated goat anti-rabbit, rabbit anti-goat or rabbit anti-mouse antibodies (Zymed
Laboratories Inc, South San Fransisco, CA). Enhanced chemiluminescence (ECL) system
(Amersham Biosciences) was used for detection.
47
4.10.1 Antibodies (I-IV)
The antibodies used in immunoblot analysis were rabbit polyclonal anti-cytochrome c (Clontech)
(I,II,III), polyclonal anti-Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA) (I), mouse
monoclonal anti-Bad (Transduction Laboratories, Lexington, KY) (I), mouse monoclonal anti-Bax
(Transduction Laboratories) (I), rabbit polyclonal anti-Bcl-2 (Santa Cruz) (I), goat polyclonal anti-
Akt1/2 (Santa Cruz) (I), rabbit polyclonal anti-phospho-Akt (Ser-473) (Santa Cruz) (I), rabbit
polyclonal anti-Bim (Sigma-Aldrich) (I,II), mouse monoclonal anti-caspase-8 (Cell Signaling
technology, Beverly, USA) (III), rabbit polyclonal anti-caspase-9 (H-83) (Santa Cruz) (III), mouse
monoclonal anti-Bcl-XL (2H12) (Zymed laboratories Inc., CA, USA) (III, IV), rabbit polyclonal
anti-Bid (Cell signaling technology) (III), rabbit polyclonal anti-FLIP S/L (H-202) (Santa Cruz)
(IV), rabbit polyclonal anti-XIAP (ProSci Inc., Poway, CA) (IV), rabbit polyclonal anti-cIAP
(ProSci Inc.) (IV), rabbit polyclonal anti-phospho-GSK3a/b (Ser21/9) (Cell Signaling technology)
(II), rabbit polyclonal anti-caspase-3 (Santa Cruz), mouse monoclonal anti-cyclin D3 (BD
Transduction Laboratories), mouse monoclonal Kip1/p27 (BD Transduction Laboratories), mouse
monoclonal anti-COX4 (Clontech) (II), rabbit polyclonal anti-actin (SantaCruz) (II, III),
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.), HRP-
rabbit anti-goat IgG (Zymed Laboratories Inc.) and HRP-goat anti-mouse IgG (Zymed Laboratories
Inc.).
4.11 RT-PCR (IV)
Briefly 1x106 cells were washed twice with cold PBS and the pellets were stored at -70 C prior to
analysis. Total mRNA extraction was done by using MasterPureTM RNA purification Kit (Epicentre
Technologies, WI) according to manufacturer’s instructions. Total mRNA amounts were measured
using spectrophotometry (GeneQuant, Pharmacia).
For cDNA sythesis 2 µg mRNA, 40 pmol p(dT)15 and RNA water ad 18 µl were mixed and
incubated at 65 C for 5 min. After incubation 1 X RT buffer (5 X M-MLV RT buffer, Promega),
10 mM each dNTP and 8 U of RT (M-MLV RT, Promega) were added to reaction mixture (final
volume 25 µl). cDNA was synthesized at 45 C for 1 h. After synthesis 1:1 TE buffer was added
and samples were boiled for 3 min. cDNA synthesis was controlled by PCR using 2µ-globulin
primers (sense 5'-CCA-GCA-GAG-AAT-GGA-AAG-TC-3', anti-sense 5'-GAT-GCT-GCT-TAC-
ATG-TCT-CG-3'). Other primers used were as follows: FLIP-S sense 5`-CTG-GTT-GCC-CCA-
48
GAT-CAA-CTG-3`, FLIP-S anti-sense 5`CTG-GTT-GCC-CCA-GAT-CAA-CTG-3`, FLIP-L
sense 5´-GTA-TAT-CCC-AGA-TTC-TTG-GC-3`, FLIP-L anti-sense 5`GGC-TTC-CCT-GCT-
AGA-TAA-GG-3`, BCL-XL sense 5'-ATG-GCA-GCA-GTA-AAG-CAA-G-3', BCL-XL anti-sense
5'-GCT-GCA-TTG-TTC-CCA-TAG-A-3', C-IAP-1 sense 5'-GCC-ATC-TAG-TGT-TCC-AGT-
TC-3', C-IAP-1 anti-sense 5'-CAG-TGG-TAT-CTG-AAG-TTG-AC-3', C-IAP-2 sense 5'-CAA-
TTG-GGA-ACC-GAA-GGA-TA-3', C-IAP-2 anti-sense 5'-ACT-TGC-AAG-CTG-CTC-AGG-AT-
3', XIAP sense 5’-CGA-AGT-GAA-TCT-GAT-GCT-GTG-3’, XIAP anti-sense 5’-CCC-TCC-
TCC-ACA-GTG-AAA-GC-3’, survivin sense 5'-CTG-AGA-ACG-AGC-CAG-ACT-TG-3',
survivin anti-sense 5'-GCA-CTT-TCT-TCG-CAG-TTT-TC-3'. Polymerase chain reaction (PCR)
was carried out under the following conditions: 3 min at 94 C followed by 35 cycles of 1 min at 94
C, 1 min at 57 C and 1 min at 72 C with a final 10 min extension at 72 C. PCR products were
separated by agarose gel electrophoresis.
4.12 Preparation of nuclear extracts (IV)
For the preparation of nuclear extracts the cells were washed with PBS and suspended in a
hypotonic buffer (1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 mM HEPES). After 5 min
incubation on ice, 0.08% NP-40 was added and suspension was incubated further 2 min before
centrifugation (400 g, 2 min, 4 C). Pellets containing the nuclei were suspended in hypertonic
buffer (25 % glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 420 mM
NaCl, 20 mM HEPES) and incubated at 4 C for 30 min with smooth shaking. Finally, samples
were centrifuged (25 000 g, 20 min, 4 C) and supernatants were stored at -80 C. The amount of
nuclear proteins was analyzed with Lowry's method (DC protein assay, BioRad Laboratories).
4.13 EMSA assay (IV)
Double-stranded oligonucleotide containing the NF- B consensus binding (5'-AGT TGA GGG
GAC TTT CCC AGG C-3'; Promega, Madison, WI, USA) was labeled with [ -32P]ATP (3000
Ci/mmol; Amersham Pharmacia Biotech Benelux, AT Roosendal, Netherlands) using T4-
polynucleotide kinase (MBI Fermentas, Hanover, MD, USA). Labeled probe was separated using
Probe Quant G-50 micro columns (Amersham Pharmacia Biotech Benelux) prior to their use in
EMSA experiments.
49
Extracted nuclear proteins (6 µg protein per reaction) were incubated for 20 minutes with [ -32P]ATP labeled probes in binding buffer (10 % glycerol, 1 mM DTT, 1 mM EDTA, 25 mM
HEPES, 100 mM NaCl) and 1.5 µg poly(dI-dC) in final reaction volume of 20 µl. The protein-
DNA complexes were separated (25 mA, 90-120 min) on a high ionic strenght gel (6 %
acrylamide) in running buffer (50 mM Tris, 380 mM glycine, 1 mM EDTA). After electrophoresis,
the gel was dried (1h, +60 C) and exposed to autoradiography film for 1-2 days at -80 C.
4.14 Cloning (II-IV)
Plasmids coding for caspase-9, caspase-10, Bcl-XL, dominant negative-FADD, FLIP-S and FLIP-
L, were obtained by amplification of caspase-9, caspase-10, Bcl-XL, DN-FADD (lacking the death
effector domain (DED)) sequences by PCR using cDNA from HF28RA as template. Primer
sequences for amplification were for caspase-9 (S 5'-AT ATT TAA ATA GCC ACC ATG GAC
GAA GCG GAT CGG-3', AS 5'-ATA TTT AAA TGG GCC CTG GCC TTA TGA TGT-3'),
caspase-10 (S 5'-AT ATT TAA ATA GCC ACC ATG AAA TCT CAA GGT CAA C -3', AS 5'-A
TAT TTA AAT CTG CTA TAA TGA AAG TGC ATC-3'), Bcl-XL (S 5'-AT ATT TAA ATA
GCC ACC ATG TCT CAG AGC AAC GGG AGC-3', AS 5'-ATA TTT AAA TCA GTG TCT
GGT CAT TTC CGA C-3'), FADD (S 5-AT ATT TAA ATA GCC ACC ATG CGC GTC GAC
GAC TTC GAG GCG G-3’, AS 5'-AT ATT TAA ATG CCC ATC AGG ACG CTT CGG AG-
3’), FLIP-S (S 5'-ATA TTT AAA TCT AAG AGT AGG ATG TCT GCT GAA G-3', AS 5'-ATA
TTT AAA TAT GTT AAT CAC ATG GAA CAA TTT C-3') and FLIP-L (S 5'-ATA TTT AAA
TCT AAG AGT AGG ATG TCT GCT GAA G-3', AS 5'-ATA TTT AAA TGG TTT CTT ATG
TGT AGA GAG GAT AAG-3'). Swa I- restriction enzyme cleavage sites (ATTTAAAT) were
designed to the end of primers (bolded). PCR fragments were isolated from 1.2% agarose gel using
Montage kit (Millipore) and then sub-cloned into the pcr2.1-TOPO-TA vector (Invitrogen,
Carlsbad, CA) and transformed to competent One Shot TOP10-E. coli (Invitrogen).
4.15 Site-directed mutagenesis, cloning and production of lentiviruses (II-IV)
Dominant negative caspase-9 and caspase-10 were generated by introducing a point mutation to
catalytic cysteines, Cys287Ser (TGT AGT) and Cys401Ser (TGC AGC), respectively. Point
mutation was generated by megaprimer method. Megaprimers were generated by PCR using
Phusion DNA polymerase (Finnzymes, Espoo, Finland). Mutation primer for caspase-9 was S 5'-C
CAG GCC AGT GGT GGG GAG CAG-3' and for caspase-10 S 5'- C ATC CAG GCC AGC CAA
50
GGT GAA G-3' (point mutations are bolded in primer sequence) and these primers were pared with
SwaI-sites containing anti-sense primers mentioned above. PCR products were isolated from
agarose gel and used as megaprimer in second round PCR with SwaI-sites containing sense primers
mentioned above.
PCR products were isolated from agarose gel and then sub-cloned into the pcr2.1-TOPO-TA vector
and transformed to competent One Shot TOP10-E.coli. Inserts in TOPO-vectors were fully
sequenced and verified to contain the desired product. After sequencing inserts were cloned to Swa
I site of the pWPI-IRES-GFP (TronoLab, Switzerland) lentivirus vector. The lentiviral vectors
containing DN-caspase-9-IRES-GFP, DN-caspase-10-IRES-GFP, DN-FADD-IRES-GFP and Bcl-
XL-IRES-GFP were produced following the protocol described earlier [41]. IRES-GFP pWPI
lentiviruses were also produced and used as vector control. Titers of produced lentiviruses were
1.4x10E6 t.u.(transducing units)/ml (Bcl-XL), 4.3x10E6 t.u./ml (DN-casp9), 2.8x10E7 t.u./ml (DN-
casp10), 8.3x10E6 t.u./ml (DN-FADD) and 5.8x10E7 t.u./ml (vector control) as measured by
FACS analysis [41].
4.16 Lentiviral transduction, FACS analysis and sorting of GFP-positive cells (II-IV)
The HF28RA and HF1A3 cells were transduced with lentiviral vectors in 6-well plate using
100000 cells/ml/well, in the presence of polybrene 8 g/ml (SigmaAldrich, Stenheim, Germany)
and with 1 t.u./cell. After three days, cells were collected and cultured in fresh medium and the
amount of GFP-positive cells were analyzed by FACScan flow cytometer and after expanding the
cells, the transduced cells were further enriched and purified over 96 % pure population, according
to the GFP expression with cell sorting using EPICS Elite ESP flow cytometer (Beckman Coulter,
Fullerton, CA, USA).
51
5. RESULTS AND DISCUSSION
5.1 Dexamethasone induces cell cycle arrest and apoptosis in dose-dependent manner
(unpublished data)
Glucocorticoids (GCs) have been shown to induce cell cycle arrest and apoptosis in lymphoid cells
(Ausserlechner et al., 2004; Gaynon and Carrel, 1999; King and Cidlowski, 1998; Renner et al.,
2003). Synthetic glucocorticoid, dexamethasone (Dex), induced cell cycle arrest and apoptosis in a
dose-dependent manner in HF28RA cells (Fig.8). Based on thymidine incorporation, proliferation
of HF28RA cells decreased considerably at the concentration of 0.01 µM (Fig.8A) whereas
apoptosis was induced at ten-time higher concentration of Dex as demonstrated by flow cytometric
analysis after PI-staining (Fig.8B). The decrease in the proliferation after Dex (0.01 µM) treatment
was not due to the apoptotic cell death, but rather due to the induction of cell cycle arrest at G0/G1
(Fig.8C).
Furthermore, HF28RA cells induced to G0/G1 cell cycle arrest for 24 h could enter back to the
cycle within the next 24 h (data not shown). Other studies have also indicated that anti-proliferative
effects of GCs may be mediated by a reversible G1-block in cell cycle progression since after
release of Dex, cells reinitiated cell cycle progression and entered S-phase (Glick et al., 2000; Goya
et al., 1993; Sanchez et al., 1993). In addition, prolonged induction of cell cycle arrest (48 hours)
did not lead to apoptosis and overexpression of Bcl-XL prevented Dex-induced apoptosis
(discussed later, II, Fig.1) but not cell cycle arrest in HF28RA cells (data not shown). It was also
shown in human ALL cells that Bcl-2 protected cells against GC-induced apoptosis but did not
affect GC-mediated growth arrest separating the anti-proliferative and apoptosis-inducing effects of
GCs (Hartmann et al., 1999). Thus, our finding supports the previous suggestion that cell cycle
arrest and apoptosis are not directly connected to each other, although it has been thought to be the
case in highly proliferating cancer cells (King and Cidlowski, 1998).
52
A) B)
C)Control Dex 0.01 µM
% of cells Control Dex 0.01µMHypodiploid 4.08 8.74G0/G1 56.52 84.94S/G2/M 39.11 6.24
Figure 8. Dex induces apoptosis and cell cycle arrest in a dose dependent manner in HF28RA
cells. (A) Cells were treated with indicated concentrations of Dex. After 20 h of incubation, 3µCi
of (3H) thymidine was added to cells for 4 h. Cells were harvested and the incorporated
radioactivity was measured by beta scintillation counting. (B,C) Cells were incubated in indicated
concentrations of Dex. After 24 h of incubation, cells were harvested for cell cycle analysis.
Hypodiploid cells (apoptotic cells), cells at G0/G1 and cells at S/G2/M are indicated as histogram
regions M1, M2 and M3, respectively. The proportion of cells at different stages of cell cycle is
shown under the histograms.
Cell cycle progression is controlled by cyclin-dependent kinases (CDKs) and their regulatory
subunits, cyclins. The activity of cyclin D-dependent kinases CDK4 and CDK6 is tightly regulated
53
by inhibitors, including p27Kip. They halt cell cycle at G1 phase, by binding to, and inactivating,
cyclin-CDK complexes. Active CDK4/6 complexes phosphorylate the retinoblastoma protein (Rb).
The hyperphosphorylation of Rb leads to the release and activation of E2F, the transcription factor
essential for the progression of cell cycle from G1 to S (Ausserlechner et al., 2004). Cell cycle
regulators shown to be modulated by GCs include cyclin D3, c-myc, CDK4 and CDK6, which have
been shown to be downregulated as well as p21Waf1 and p27Kip which have been shown to be
upregulated (Ausserlechner et al., 2004; Greenstein et al., 2002). Our results also showed that Dex-
induced downregulation of cyclin D3 in 8h and slight upregulation of p27Kip in 16 h as
demonstrated by Western blotting (Fig.9).
Dex 1 h
Dex 4 h
Dex 8 h
Dex16 h
Control 16 h
CyclinD3
Control 1 h
p27Kip
Figure 9. Dexamethasone decreases the amount of cyclin D3 protein and slightly increases the
amount of p27Kip. Cells were treated with 0.01 µM Dex for 1, 4, 8 and 16 h and the amount of
cyclin D3 and p27Kip proteins were detected by Western blotting.
5.2 Mechanism of Dex-induced apoptosis (I, II)
GCs are frequently used in the treatment of various lymphoid malignancies, including leukemia,
lymphoma and multiple myeloma. Despite their extensive clinical use, the molecular mechanisms
leading to GC-induced apoptosis are not fully understood. In order to better understand the
molecular mechanisms of Dex-induced apoptosis we analyzed the kinetics and the signaling
requirements of Dex-induced apoptosis in a human follicular lymphoma cell line, HF28RA. Dex
induced apoptosis in a delayed manner. The apoptotic changes including the loss of mitochondrial
membrane potential ( m), release of cyt c from mitochondria to cytosol, activation of caspase-3
(measured as protease activity against a fluorogenic DEVD peptide) and DNA fragmentation were
induced after 12 h stimulation with Dex (I, Fig.1 and 6C). Kinetics of caspase-3 activation was also
analyzed by Western blotting and similar results were obtained (Fig.10). Caspase-8 was also
activated as demonstrated by Western blotting (Fig.10).
54
Involvement of mitochondrial changes in Dex-mediated apoptosis has also been demonstrated in
several other studies with many cell types, including lymphoid cells (Camilleri-Broet et al., 1998;
Castedo et al., 1995; Herr et al., 2007). In addition, we showed that overexpression of Bcl-XL
completely prevented Dex-induced apoptotic changes indicating that mitochondrial activation is
necessary for Dex-induced apoptosis (II, Fig.1C, D). Other studies have also shown that
overexpression of anti-apoptotic Bcl-2 family proteins protects against GC-induced apoptosis
(Alnemri et al., 1992; Caron-Leslie et al., 1994; Hartmann et al., 1999; Smets et al., 1999). In
addition, GC-induced cell death was enhanced in thymocytes from Bcl-2 knockout mice (Veis et
al., 1993).
It is commonly thought that MOMP results in the release of cyt c from mitochondria leading to the
formation of apoptosome and subsequent activation of caspase-9. Therefore, it is not surprising that
overexpression of dominant negative (DN) caspase-9 prevented Dex-induced apoptosis (II, Fig.1E).
Other studies have also shown that caspase-9 is necessary for GC-induced apoptosis (Hakem et al.,
1998; Kuida et al., 1998; Planey et al., 2003). Interestingly, DN caspase-9 prevented also the loss
of m (II, Fig.1F) indicating that caspase-9 is acting upstream of or even within mitochondria or
serves an essential amplification loop back to mitochondria. Previous studies have also shown that
caspase-9 regulates mitochondrial changes. It was shown that caspase-9 deficiency in caspase-9
knockout mice prevented Dex-induced loss of mitochondrial membrane potential (Hakem et al.,
1998; Kuida et al., 1998). In addition, the rituximab-induced release of cytochrome c and loss of
mitochondrial membrane potential were regulated by caspase-9 in FL cells (Eeva et al., 2009). It
has also been shown in caspase-3 and caspase-7 double knockout mice that caspases 3 and 7 are
critical mediators of mitochondrial events of apoptosis (Lakhani et al., 2006). The detailed
mechanisms how caspases regulate mitochondrial events are not known but I would like to
speculate that there is an essential amplification loop from caspase-9 back to mitochondria via
caspase-3(/7) and caspase-8 since activation of caspase-8 was also induced by Dex (Fig.10).
Recently, it was shown that cathepsin B was released from lysosomes to the cytosol in thymocytes
after GC treatment (Wang et al., 2006). It was suggested that caspase-9 activates directly caspase-3
and also indirectly through lysosomal amplification loop by activating the release of cathepsin B
from lysosomes to cytosol leading to activation of caspase-8 and subsequently activation of
caspase-3 (Wang et al., 2006). Future studies will show whether there is co-operation of
mitochondrial and lysosomal death pathways during Dex-induced apoptosis in HF28RA cells.
55
It has been previously reported that Dex-induced apoptosis is countered by protein synthesis
inhibitors (Cifone et al., 1999; Lemaire et al., 1999). The slow kinetics of Dex-induced apoptosis
offers plenty of time for protein synthesis to occur. Indeed, in our experiments, treatment of
HF28RA cells with protein synthesis inhibitor, cycloheximide (CHX), prevented partly Dex-
induced apoptosis in a dose dependent manner (I, Fig.2A), indicating that new protein synthesis is
required for the induction of apoptosis. In addition, our results showed that protein synthesis is
required already before or at mitochondrial stage of apoptosis (I, Fig.2B). However, the inability of
CHX to completely prevent Dex-induced apoptosis indicates that protein synthesis independent
apoptotic mechanisms could also be involved. However, more probable explanation could be that
the amount of CHX needed to prevent Dex-induced protein synthesis becomes toxic itself.
The Bcl-2 family member consistently shown to be upregulated by glucocorticoids in lymphoid
cells is Bim (Abrams et al., 2004; Bachmann et al., 2005; Planey et al., 2003; Wang et al., 2003;
Zhang and Insel, 2004). We showed that Dex induced upregulation of all three major isoforms of
Bim in HF28RA cells. Upregulation of Bim was shown from total cell lysates (I, Fig.2C) and also
from mitochondrial fraction of cellular proteins (I, Fig.7). The three major isoforms of Bim
(BimEL, BimL and BimS) are generated by alternative splicing and all of them induce apoptosis.
The kinetics of the apoptotic events correlated with the upregulation of the Bim protein indicating
that Bim plays a central role in Dex-induced apoptosis in HF28RA cells.
The ratio of proapoptotic to anti-apoptotic Bcl-2 family members is thought to be critical in
determining whether the cell will undergo apoptosis. HF28RA cells, as most cases of follicular
lymphoma (Weiss et al., 1987), have a t(14;18) translocation, which causes over-expression of the
bcl-2 gene (Eray et al., 2003). In some cases a high level of Bcl-2 expression has been shown to
delay, but not to prevent GC-induced apoptosis (Hartmann et al., 1999; Strasser et al., 1991;
Strasser et al., 1995). Therefore, one explanation for slow kinetics of Dex-induced apoptosis could
be that accumulation of Bim protein in mitochondria is required to antagonize first the anti-
apoptotic Bcl-2 (and also other anti-apoptotic Bcl-2 family members) before Bax and/or Bak are
activated to induce apoptosis.
5.3 Regulation of dexamethasone-induced apoptosis by PI3-kinase-Akt pathway (I)
PI3-kinase-Akt-pathway is activated in a wide variety of cancers which results in enhanced
resistance to apoptosis through multiple mechanisms. It has been shown that inhibition of PI3-
kinase decreases cell survival and enhances the effects of chemotherapeutic drugs in many types of
56
cancer cells (Asselin et al., 2001; Clark et al., 2002; Hu et al., 2002; Ng et al., 2000; Wang et al.,
2002). We showed that Akt is constitutively phosphorylated in HF28RA cells (I, Fig.4C).
Inhibition of PI3-kinase-Akt pathway by LY294002 and Akt inhibitor completely blocked the
phosphorylation and enhanced considerably the sensitivity of HF28RA cells to dexamethasone (I,
Fig.4). Interestingly, apoptosis proceeded with accelerated kinetics, indicating that active PI3-
kinase-Akt-pathway does not only protect from but also delays Dex-induced apoptosis (I, Fig.5).
Dex-induced activation of caspase-3 and -8 was also enhanced and accelerated in the presence of
LY294002 (Fig.10, I, Fig.6B). In spite of the importance of PI3-kinase-Akt as a survival-promoting
pathway, treatment with inhibitors alone did not cause cell death, in contrast to the results obtained
in some other cell models (Liu et al., 2001; Xu et al., 2003).
Dex 0.1µMLY 10 µM
- + - + - + - + - + - + - + - +- - + + - - + + - - + + - - + +
pro-caspase8
cleaved caspase8
cleaved caspase-3
4h 8h 16h 24h
pro-caspase3
Figure 10. PI3-kinase inhibitor, LY294002, enhances and accelerates Dex-induced activation of
caspase-3 and -8. HF28RA cells were pre-incubated with 10 µM LY294002 for 1 h prior to the
addition of 0.1 µM Dex for 4, 8, 16 and 24 h. After incubations, proteins were isolated and
cleavage of caspase-3 and -8 was analyzed by Western blotting.
Furthermore, inhibition of PI3-kinase with LY294002 markedly enhanced Dex-induced apoptosis
already at the mitochondrial level (I, Fig.6A, C). Previous findings support our results since it has
been shown that Akt inhibited apoptosis at a pre-mitochondrial level inhibiting the cytochrome c
release and the loss of mitochondrial membrane potential (Gottlob et al., 2001; Kennedy et al.,
1999). One connection between Akt and mitochondria is the phosphorylation of the pro-apoptotic
Bcl-2 family protein Bad. Bad is phosphorylated at Ser-136 by Akt and also at Ser-112 and at Ser-
155 by other kinases. Bad does not contain a mitochondrial targeting sequence but localizes to
mitochondria, in a phosphorylation-dependent manner. When Bad is phosphorylated it binds to 14-
3-3 proteins in the cytosol. Dephosphorylated Bad is released from 14-3-3 and becomes free to
57
heterodimerize with anti-apoptotic members of Bcl-2 family proteins (Bcl-2, Bcl-XL and Bcl-w) in
mitochondria, thereby inhibiting their anti-apoptotic activity (Gross et al., 1999). Our results
showed that inhibition of PI3-kinase induced translocation of Bad to mitochondria (I, Fig.7).
Inhibition of PI3-kinase-Akt pathway did not alone induce apoptosis indicating that translocation of
Bad to mitochondria itself is not enough to induce apoptosis but another apoptotic signal is
required. It has been reported that Bim can bind not only to anti-apoptotic Bcl-2 family members
but also to Bax and/or Bak (Huang and Strasser, 2000; Letai et al., 2002; Marani et al., 2002). In
contrast, Bad has been reported to bind only Bcl-2 and its homologues but not to Bax or Bak (Letai
et al., 2002). Accordingly, it has been postulated that Bim is a direct inducer of apoptosis, whereas
Bad sensitizes cells to death stimuli by reducing the level of free anti-apoptotic Bcl-2 family
proteins. To investigate the possible partners of Bad and Bim in mitochondria, the subcellular
localization of Bax, Bcl-XL and Bcl-2 was analyzed. In agreement with previously published
results, anti-apoptotic proteins Bcl-2 and Bcl-XL were predominantly present in mitochondria (I,
Fig.7). The pro-apoptotic member of the Bcl-2 family, Bax, is thought to reside in the cytosol of
healthy cells and in response to apoptotic stimuli, Bax undergoes a conformational change that
leads to the translocation of Bax to mitochondria (Gross et al., 1998; Nechushtan et al., 1999).
However, it has been also shown that Bax can be loosely associated with the outer mitochondrial
membrane when not activated. Our results showed that Bax was predominantly present in
mitochondria already in untreated HF28RA cells (I, Fig.7). In conclusion, based on our results and
the current knowledge of Bim and Bad action, it seems that Bad is a sensitizer of apoptosis and
other events induced by Dex, such as upregulation of Bim protein expression, are required to
trigger apoptosis. It seems that translocation of Bad potentiates apoptosis by binding to anti-
apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) freeing Bax and Bim to interact with each other and
thereby leading to activation of Bax.
Akt can also regulate cell survival by transcription-based mechanisms. Members of the forkhead
family of transcription factors have been shown to be direct targets of Akt. Phosphorylation of
forkhead proteins by Akt appears to alter their subcellular localization. In the absence of Akt
activation, forkhead proteins are predominantly present in the nucleus where they are able to
promote transcription of pro-apoptotic target genes (Nicholson and Anderson, 2002). It has been
shown that PI3-kinase-Akt signaling inhibits Bim expression by phosphorylating the FOXO3
(forkhead box O3A; also known as FKHRL1) (Dijkers et al., 2002; Strasser, 2005). Therefore, we
analyzed the effect of PI3-kinase inhibitor LY294002 on the expression of Bim. However,
58
inhibition of PI3-kinase had no effect on Bim expression in 24 h (I, Fig 7). In addition, further
kinetic study showed similar results (Fig.11).
Dex 0.1µMLY 10 µM
- + - + - + - + - + - + - + - +- - + + - - + + - - + + - - + +
4h 8h 16h 24h
Bim ELBim LBim S
Figure 11. PI3-kinase inhibitor, LY294002, has no effect on Dex-induced upregulation of Bim.
HF28RA cells were pre-incubated with 10 µM LY294002 for 1 h prior to the addition of 0.1 µM
Dex for 4, 8, 16 and 24 h. After incubations, proteins were isolated and the amounts of Bim
proteins were analyzed by Western blotting.
5.4 The role of glycogen synthase kinase-3 (GSK3) in Dex-induced apoptosis (II)
GSK3 has been shown to promote the intrinsic apoptotic signaling induced by many different
stimuli. GSK-3 facilitates the intrinsic apoptotic signaling through targeting several proteins that
regulate signals leading to disruption of mitochondria (Beurel and Jope, 2006). Activity of GSK3 is
negatively regulated through phosphorylation by Akt on Ser-21 in GSK3- or Ser-9 in GSK3- .
However, several other kinases have also been shown to phosphorylate these inhibitory sites. The
involvement of GSK3 in Dex-induced apoptosis was examined by using two structurally different
GSK3 inhibitors, LiCl and SB-216763. Although both of the inhibitors inhibit also other targets,
the only common target is GSK3 (Cohen and Goedert, 2004). To confirm that GSK is inhibited
with LiCl and SB-216763, the inhibitory phosphorylation status of both isoforms of GSK3 was
studied. Interestingly, the inhibitory phosphorylations by these inhibitors were dependent on
subcellular localization of GSK3 / since the amount of phosphorylated GSK3 and GSK3 (at
inhibitory site) increased in cytosol in SB-216763- treated cells and in mitochondria in LiCl-treated
cells (II, Fig.2D). It has been previously shown that lithium increases the Ser-9 phosphorylation of
GSK3 (Zhang et al., 2003). It has been also shown that lithium induces activation of Akt (Tajes et
al., 2009). In addition, following PI3-kinase activation, Akt was shown to translocate to the
mitochondria and phosphorylate Ser-9 of mitochondrial GSK3 (Bijur and Jope, 2003) Therefore,
one mechanism how LiCl might increase inhibitory phosphorylations of GSK3 / at mitochondria
could be that LiCl activates Akt which subsequently translocates to mitochondria and
59
phosphorylates mitochondrial GSK3 / at inhibitory serine residues. In contrast to our results, SB-
216763 which acts as an ATP competitor has been shown to inhibit GSK3 but not cause increase in
Ser21/9 phosphorylation (Zhang, 2003).
Furthermore, we showed that both inhibitors of GSK3 attenuated Dex-induced up-regulation of
Bim, loss of mitochondrial membrane potential, release of cyt c and DNA fragmentation (II, Fig.2).
These results indicate that GSK-3 contributes to Dex-induced apoptosis by regulating the
expression of Bim. It has also been shown that GSK3 is required for trophic deprivation-induced
expression of Bim in neurons (Hongisto et al., 2003). It has also been shown in osteoblast cells, that
glucocorticoids induce apoptosis through the activation of GSK3 (Yun et al., 2009). However, to
date, there are no previous studies demonstrating the involvement of GSK3 in the Dex-induced up-
regulation of Bim.
FOXO transcription factors regulate the expression of Bim (as discussed earlier). However, it has
been shown that Bim induction requires simultaneous activation of three different death signaling
pathways (FOXO, Mybs, c-Jun) in neurons (Biswas et al., 2007). In future we will study whether
FOXO transcription factors are responsible for Dex-induced upregulation of Bim, and how GSK3
contributes to Bim regulation. It would be of great interest also to see whether PI3-kinase-Akt
pathway controls GSK3 activity in HF28RA cells.
In contrast to our results, it has been shown that GSK3 has also inhibitory effect on glucocorticoid-
induced apoptosis. It has been recently shown that GSK-3 -mediated GR phosphorylation inhibited
glucocorticoid-dependent NF-kappaB transrepression and attenuated the glucocorticoid-dependent
cell death of osteoblasts (Galliher-Beckley et al., 2008). Additionally, it has been shown that
GSK3-inhibitor (TDZD) induced apoptosis in myeloma cell lines and TDZD-mediated inhibition of
GSK3 resulted in dephosphorylation and activation of FOXO3a (Zhou et al., 2008).
5.5 Molecular mechanisms of TRAIL-induced apoptosis (III)
TRAIL induces apoptosis through engaging TRAIL-R4 (DR4) and/or TRAIL-R5 (DR5). Both
receptors are expressed normally simultaneously on the same cell. To investigate the expression of
TRAIL-R1 and TRAIL-R2 on HF28RA and HF1A3 cell lines, cells were stained with Abs against
the receptors. FACS analysis showed that both cell lines expressed both receptors on the cell
surface (Fig.12A, B).
In spite of the fact that both receptors are expressed on the same cell, generally TRAIL-R2 seems
to play a more important role than TRAIL-R1 in triggering apoptosis (Kelley et al., 2005).
60
Antibody inhibition study was done to resolve, whether both TRAIL receptors mediate apoptosis in
HF28RA and HF1A3 cell lines. Both blocking Abs (TRAIL-R1 or TRAIL-R2 mAb) decreased
TRAIL-induced apoptosis indicating that both of the receptors were capable of mediating apoptosis
in HF28RA and HF1A3 cells. Simultaneous TRAIL-R1 and TRAIL-R2 blockade almost
completely prevented TRAIL-induced apoptosis in both cell lines (Fig.12C).
Cou
nts Anti-mouse PE
TRAIL-R1+anti-mouse PE
TRAIL-R2+anti-mouse PE
PE
HF1A3
Cou
nts
PE
Anti-mouse PETRAIL-R1+anti-mouse PE
TRAIL-R2+anti-mouse PE
HF28RA
A) B)
Figure 12. Functional expression of TRAIL-receptors on HF28RA and HF1A3 cells. A-B)
HF28RA and HF1A3 cells were stained with mAbs against TRAIL-R1 and TRAIL-R2 followed by
PE-labeled goat anti-mouse. Isotype matched mouse IgG1 was used as control. Stained cells were
analyzed by flow cytometry and histograms of the FACS analysis are presented. C) Cells were
incubated with TRAIL-R1 and/or TRAIL-R2 mAb for 30 min to block either one or both of the
receptors, prior to addition of TRAIL. After 24 h, the percentage of apoptotic cells was analyzed by
propidium iodide staining. The data are presented as a mean + SEM from three independent
experiments.
61
To study whether caspase-8 is required for TRAIL-induced apoptosis, cells were pre-incubated
with 15 µM caspase-8 inhibitor, Z-IETD-FMK for 1 hour prior addition of TRAIL for 24 h. As
expected, Z-IETD-FMK completely prevented TRAIL-induced apoptosis in both cell lines (Fig.
13).
Figure 13. Inhibition of caspase-8 prevents TRAIL-induced apoptosis. Cells were pre-incubated
with 15 µM caspase-8 inhibitor, Z-IETD-FMK for 1 hour prior to addition of TRAIL for 24 h. The
percentage of apoptotic cells was analyzed by propidium iodide staining. The data are presented as
a mean + SEM from three independent experiments.
Besides caspase-8, caspase-10 can also be recruited to the TRAIL DISC (Kischkel et al., 2001).
However, the importance of caspase-10 in induction of apoptosis is controversial (Kischkel et al.,
2001; Sprick et al., 2002). To investigate the potential role of caspase-10 during TRAIL-induced
apoptosis, cells were transduced with dominant negative (DN) form of caspase-10 in IRES-GFP-
lentivector. Overexpression of DN-caspase-10 was confirmed by Western blotting (Fig. 14A).
HF28RA and HF1A3 cells transduced with "empty" IRES-GFP lentivirus vector were used as
controls (HF28RA GFP, HF1A3 GFP). In HF1A3 cells, caspase-10 is not involved in TRAIL-
induced apoptosis as the DN form of caspase-10 had no effect on TRAIL-induced apoptosis
(Fig.14B). In contrast, in HF28RA cells, DN caspase-10 decreased TRAIL-induced apoptosis
indicating that caspase-10 may play a minor role (Fig.14B).
62
HF1A3
GFPDN-casp10
HF28RA
GFPDN-casp10
A)
Figure 14. The role of caspase-10 in TRAIL-induced apoptosis. A) Total cellular proteins were
isolated from vector control (GFP expressing) cells and dominant- negative caspase-10 expressing
cells and western blot analysis was performed to confirm the overexpression of DN-caspase-10. B)
Cells were treated with 50 ng/ml TRAIL for 24 h and percentage of apoptotic cells was analyzed by
propidium iodide staining. The data are presented as a mean + SEM from three independent
experiments.
5.5.1 Type I and type II TRAIL signaling (III)
Cells can be divided into two types according to their requirement for mitochondria in death
receptor induced apoptosis. It has been suggested that in type I cells apoptosis can proceed
independently of mitochondria. In type II cells, apoptosis is thought to depend on mitochondria and
is blocked by overexpression of Bcl-2 or Bcl-XL (Scaffidi et al., 1998). In TRAIL-signaling both
apoptotic pathways have also been described. It has been shown that overexpression of Bcl-2 or
Bcl-XL does not block TRAIL-induced apoptosis in some cancer cell lines (Fulda et al., 2002;
Keogh et al., 2000; Kim et al., 2001; Rudner et al., 2001; Walczak et al., 2000). In contrast,
overexpression of Bcl-2 or Bcl-XL blocks or reduces TRAIL-induced apoptosis in other cancer cell
lines (Fulda et al., 2002; Hinz et al., 2000; Munshi et al., 2001; Rokhlin et al., 2001; Ruiz de
Almodovar et al., 2001; Srinivasula et al., 2000; Sun et al., 2001). We showed that mitochondria
have an important but distinct role during TRAIL-induced apoptosis in FL cell lines. Mitochondrial
changes (loss of m and release of cyt c) were faster and more strongly activated in HF28RA
63
cells than in HF1A3 cells while DNA fragmentation followed similar quantity and kinetics (III,
Fig.1). In addition, Bcl-XL overexpression studies revealed that in HF28RA cells TRAIL-induced
apoptosis is completely dependent on the mitochondrial activation (III, Fig.1). Recently the
requirement of mitochondrial pathway in type II cells was challenged as it was shown that
protective effect of Bcl-2 was limited to lower concentrations of TRAIL or early observation time
points (Rudner et al., 2005). To study whether increasing amount of TRAIL or duration of TRAIL
stimulus, could affect the mitochondrial dependence, cells were incubated with 50 ng/ml or 100
ng/ml TRAIL for 24 and 48 hours. The percentage of cells with depolarized mitochondrial
membrane and the percentage of apoptotic cells were measured by TMRM- and PI-stainings,
respectively. The results show that higher concentration of TRAIL or prolonged incubation time
did not change type II signaling to type I signaling, as the Bcl-XL still prevented all TRAIL-
induced apoptotic changes in HF28RA-cells (Fig.15A, B).
Figure 15. Increasing concentration of TRAIL and duration of TRAIL stimulus has no effect on
type II behavior. Cells were incubated with 50 ng/ml or 100 ng/ml TRAIL for 24 or 48 hours and
(A) percentage of apoptotic cells was analyzed by propidium iodide staining and (B) the percentage
of cells with depolarized mitochondrial membrane was analyzed by TMRM staining.
64
In contrast, in HF1A3 cells, although overexpression of Bcl-XL almost completely blocked
depolarization of mitochondrial membrane, it only decreased DNA fragmentation (III, Fig.1E, G).
Interestingly, in Bcl-XL overexpressing HF1A3 cells TRAIL-induced apoptosis was decreased
much lesser extent in 4 h than at later time points (8h and 16h) (III, Fig.1) indicating that
mitochondria might serve a late amplification loop to caspase activation in HF1A3 cells. In
conclusion, due to these differences in mitochondrial involvement during TRAIL-induced
apoptosis, we suggest that HF28RA cells represent type II cells and HF1A3 cells resemble more
type I cells. These differences in mitochondrial activation could not be explained by different
amount of Bcl-2 or Bcl-XL proteins because it has been previously shown that these cell lines
express comparable levels of Bcl-2 (Skommer et al., 2006) and we showed that amount of Bcl-XL
was even slightly higher in HF1A3 cells than in HF28RA cells (III, Fig.1A).
Interestingly, overexpression of Bcl-XL failed to prevent TRAIL-induced release of mitochondrial
cyt c to the cytosol in HF1A3 cells even the loss of mitochondrial membrane potential was
prevented (III, Fig.1E, F) indicating that these are independent events. In contrast, release of cyt c
was associated with the loss of m in HF28RA cells. Generally, the release of apoptotic proteins
from mitochondria, such as cyt c, is thought to be associated with the loss of m. However, it has
been shown that release of cyt c is independent of mitochondrial depolarization (Bossy-Wetzel et
al., 1998) and that m can still be maintained after the release of cyt c to cytosol (Waterhouse et
al., 2001). It was recently shown that also in death receptor induced (Fas and TRAIL) apoptosis
depolarization of mitochondrial membrane potential can be uncoupled from cyt c release (Samraj et
al., 2006).
It is known that mitochondria are activated in both cell types, however it is not yet understood why
type II cells exclusively utilize mitochondrial pathway in death receptor mediated apoptosis. It has
been suggested that the amount of activated caspase-8 generated at the DISC may be one critical
factor (Scaffidi et al., 1998). Our results show that large amount of caspase-8 was activated in both
cell lines after TRAIL treatment (III, Fig.2). It has been previously shown that overexpression of
Bcl-XL or Bcl-2 blocks activation of caspase-8 in type II cells but not in type I cells (Scaffidi et al.,
1998). In addition, in Jurkat cells (type II) overexpression of Bcl-XL inhibited Fas-induced
apoptosis and also caspase-8 activation (Zhao et al., 2007). Our data showed that Bcl-XL did not
prevent or even reduce the activation of caspase-8 in either of cell types (III, Fig.2). In addition,
Bid was cleaved equally in both cell lines and overexpression of Bcl-XL did not prevent cleavage of
Bid (III, Fig.2). Therefore, it seems unlikely that neither the amount of caspase-8 activation at the
DISC nor the Bid cleavage can define whether cells are type I or type II cells, at least in our model.
65
On the contrary to our result, it has been shown in glioma cells that enforced overexpression of
caspase-8 in type II cells overcomes Bcl-2 inhibition of Fas- and TRAIL-induced apoptosis,
converting cells from type II to type I (Knight et al., 2004). In addition, it was shown that
hepatocytes (type II cells) deficient in Bid were resistant to Fas-induced apoptosis and high dose of
hepatocyte growth factor (HGF) sensitized Bid deficient cells to Fas indicating a decrease in
mitochondria requirement (Zhao et al., 2007).
It is commonly thought that release of cyt c leads to the formation of apoptosome and activation of
caspase-9. However, there are not much data about the role of caspase-9 in TRAIL-induced
apoptosis. In type II human hepatocytes and colon carcinoma cells it has been previously shown
that caspase-9 inhibitor completely blocked TRAIL-induced apoptosis (Ozoren et al., 2000). In
contrast, in colon cancer cells caspase-9 inhibition failed to block TRAIL-induced apoptosis
(Ozoren et al., 2000). We show that overexpression of DN caspase-9 had no effect on TRAIL-
induced apoptosis in HF1A3 cells (III, Fig.3E-G) despite the fact that cyt c was released. In
contrast, in HF28RA cells DN caspase-9 decreased TRAIL-induced DNA fragmentation, but
surprisingly also mitochondrial changes (III, Fig.3B-D), even though caspase-9 is thought to locate
downstream from mitochondria in the apoptotic cascade. These results indicate that there might be
a late amplification loop to mitochondria through caspase-9.
Interestingly, DN-caspase-9 failed to completely prevent TRAIL-induced apoptosis in HF28RA
cells (III, Fig.3B-D) indicating that other factors, besides cyt c, could be released from
mitochondria. Indeed, the activation of mitochondrial apoptotic activity may also result in the
release of several other pro-apoptotic factors, such as Smac/DIABLO, HtrA2/Omi, AIF and
EndoG. The sensitivity of HF28RA cells to TRAIL apoptosis in spite of overexpression of DN
caspase-9 is, therefore, not surprising. It has been previously shown that TRAIL-induced apoptosis
requires Bax-dependent mitochondrial release of Smac/DIABLO (Deng et al., 2002). We could not
detect the release of mitochondrial Smac/DIABLO during TRAIL-induced apoptosis in HF28RA
and HF1A3 cells (data not shown). Nevertheless, it seems likely that in type II cells, release of a
pro-apoptotic protein(s) from mitochondria, other than cyt c or Smac/DIABLO, is/are required
before caspases can be fully activated.
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Table 1. Effect of Bcl-XL or DN-caspase-9 overexpression on TRAIL-induced apoptotic changes
in HF28RA and HF1A3 cells.
Effect of overexpression ofBcl-XL on
HF28RA (type II)
HF1A3 (type I-like)
caspase-8 activation -/+ -/+cleavage of Bid -/+ -/+loss of m --- ---release of cyt c --- -apoptosis --- - (in 4h)
-- (in 8h)Effect of overexpression ofDN-caspase9 onloss of m - -/+release of cyt c - (in 8h) -/+apoptosis -- -/+
+/- no effect, - slight decrease, -- decrease, --- prevention
Furthermore, we showed that PDTC, a potent inhibitor of NF- B, enhanced TRAIL-induced
caspase-8 activation, depolarization of mitochondrial membrane and DNA fragmentation in
HF28RA cells but had no effect on HF1A3 cells (III, Fig.4). Interestingly, in HF28RA cells
overexpression of Bcl-XL failed to prevent TRAIL-induced DNA fragmentation in the presence of
PDTC even the mitochondrial membrane depolarization (III, Fig.4A, B) and release of cyt c
(Fig.16) were still blocked. Dominant negative FADD prevented TRAIL-induced loss of
mitochondrial membrane potential and apoptosis in the absence and presence of PDTC, proving
that the death signal was still originated from the TRAIL receptor when PDTC and TRAIL were
used in combination (III, Fig.4E, F). Thus, our data show that PDTC switches TRAIL-induced
apoptotic pathway from mitochondrial dependent type II to mitochondria-independent type I
pathway in HF28RA cells. However, we showed that another more specific inhibitor of NF- B
pathway, IKK inhibitor, enhanced TRAIL-induced apoptosis in both cell types and did not switch
apoptosis to type I pathway in Bcl-XL overexpressing type II cells (III, Fig.5) indicating that NF- B
might not be the target of PDTC being responsible for the switch. Therefore, future studies are
required to solve the target of PDTC that regulates the requirement of mitochondria in TRAIL-
induced apoptosis.
In pancreatic carcinoma cells, it was shown that XIAP knockdown by RNA interference converted
Bcl-2 overexpressing type II cells to type I cells in response to TRAIL (Vogler et al., 2008). It was
also shown in childhood acute leukemia cells that small molecule XIAP inhibitors enhance TRAIL-
67
induced apoptotic changes and cooperate with TRAIL to overcome Bcl-2-mediated resistance
(Fakler et al., 2009). However, in both of these studies inhibition of XIAP in combination with
TRAIL induced loss of m and release of cyt c in Bcl-2 overexpressing cells (Fakler et al., 2009;
Vogler et al., 2008).
Furthermore, it has been shown that sublethal dose of protein synthesis inhibitor, cycloheximide
(CHX), overcomes Bcl-2 protection in type II cells in response to Fas. However, CHX induced the
loss of mitochondrial membrane potential and release of cyt c in combination with anti-Fas
(Brumatti et al., 2008). It has also been shown in TRAIL-resistant colon carcinoma cells,
proteosome inhibitors sensitizied cells to TRAIL (Nagy et al., 2006). In that study, TRAIL alone
induced partial caspase-3 activation, while the combination of TRAIL and proteosome inhibition
led to the full proteolytic activation of caspase-3. Enhanced release of mitochondrial
Smac/DIABLO to the cytosol was also shown indicating that the full activation of caspase-3 by
caspase-8 is dependent on the release of Smac/DIABLO (Nagy et al., 2006). Several other studies
have also shown that proteosome inhibitors sensitize TRAIL resistant cells to TRAIL and induce
full activation of caspase-3 (Leverkus et al., 2003; Zhu et al., 2005). We also studied the activation
of caspase-3 by Western blotting using an antibody that recognizes the cleavage products of
caspase-3. In HF28RA GFP cells (vector control cells), TRAIL induced full activation of caspase-3
which was further enhanced by PDTC. Interestingly, in Bcl-XL overexpressing cells, TRAIL
induced only partial activation of caspase-3 and the presence of PDTC led to the full activation of
caspase-3 (Fig.16).
cyt c
HF28RA GFP HF28RA Bcl-XL
Co Trail PDTC0.2 µM
Trail +PDTC Co Trail
PDTC0.2 µM
Trail +PDTC
cleavedcaspase-3
Figure 16. Effect of PDTC on TRAIL-induced release of cytochrome c and cleavage of caspase-3
in HF28RA GFP and HF28RA Bcl-XL cells. Cells were treated with 0.2 µM PDTC for 2h prior to
addition of 50 ng/ml TRAIL for additional 4h. After stimulations, cytosolic and mitochondrial
proteins were separated and the amounts of cytosolic cyt c and cleavage products of caspase-3 were
analyzed by Western blotting.
68
One hypothesis, based on the results from caspase-3 activation (Fig.16), is that Bcl-XL prevents the
release of mitochondrial protein x which is normally released to cytosol after TRAIL treatment and
required for full activation of caspase-3 probably by releasing partially processed caspase-3 from
inhibition. Furthermore, PDTC might directly or indirectly inhibit the inhibitor enabling the full
activation of caspase-3 without mitochondrial contribution (Fig.17).
Figure 17. Hypothesis of how PDTC enables type I signaling in BCL-XL overexpressing type II
cells.
5.6 The effect of microenvironmental CD40 signaling on dexamethasone-, doxorubicin- andTRAIL-induced apoptosis (IV)
Despite many different treatment approaches, the overall survival time of patients with follicular
lymphoma has not improved and it is still considered to be incurable. The progression of FL varies
between the patients, as about 15% of FL patients have a poor prognosis due to transformation to
aggressive disease and die within 3 years of diagnosis, but 20-25% may live more than 15 years
after diagnosis (Dave et al., 2004; de Jong, 2005). Recent studies have shown that the cellular
69
microenvironment influences the progression of FL (Alvaro et al., 2006; Dave et al., 2004; Glas et
al., 2005). FL cells are malignant counterparts of germinal center (GC) B cells, so like normal GC
B cells, FL cells interact with various immune cells, such as follicular helper T cells and dendritic
cells that define the tumor microenvironment (Martinez et al., 2008).
CD40 ligation (CD40 receptor on GC B cells and CD40L on helper T cells) is known to be an
important mediator of survival signals in GCs and might have an impact on treatment of follicular
lymphoma. It has been shown that CD40 stimulation rescues FL B cells from Fas-, B cell receptor
(BCR)-, rituximab- and TRAIL-induced apoptosis (Eeva et al., 2003; Eeva et al., 2007; Eeva et al.,
2009; Travert et al., 2008). We tested responses of CD40 signaling on dexamethasone- and
doxorubicin-induced apoptosis, both of which are currently used in treatment protocols of FL, as
well as on TRAIL-induced apoptosis which is in clinical trials, in three human follicular lymphoma
cell lines. The cell lines have previously been characterized according to surface marker expression
and it was shown that follicular lymphoma HF4.9 cells originate from the earlier maturation stage
of germinal center development than HF28RA and HF1A3 cells (Eray et al., 2003). Our results
showed that CD40 stimulation protected HF28RA and HF1A3 cells from dexamethasone-,
doxorubicin- and TRAIL-induced apoptosis (IV, Fig.1). On the contrary, CD40 itself induced
apoptosis in HF4.9 cells (IV, Fig.5). This result raised a question whether maturation stage of FL
cells determines the outcome of CD40 signaling. In lymphoma B cells both CD40-induced
proliferation/survival and induction of growth arrest/apoptosis have been reported (Dallman et al.,
2003). CD40 ligation has been shown to decrease the proliferation or induce apoptosis in high
grade B cell lymphomas (Funakoshi et al., 1994; Wang et al., 2008). In contrast, in low-grade B
cell malignancies CD40 has been shown to promote survival (Ghia et al., 1998; Travert et al.,
2008).
CD40 has been shown to activate multiple signaling pathways. However, signaling pathways
responsible for anti-apoptotic function are not fully understood. Many studies have shown that
CD40 induces activation of NF- B (Berberich et al., 1994; Coope et al., 2002; Homig-Holzel et al.,
2008; Lee et al., 1999; Travert et al., 2008). In B cells, NF- B is constitutively active but can be
further induced (Liu et al., 1991). Our EMSA results also showed that NF- B was constitutively
active but the activation could be strongly enhanced by CD40 (IV, Fig. 2B). Recently it was shown
that CD40 stimulation protects FL cells from TRAIL-induced apoptosis through activation of NF-
B (Travert et al., 2008). We used two different NF- B pathway inhibitors, antioxidant pyrrolidine
dithiocarbamate (PDTC) and specific I B kinase (IKK)-inhibitor (BMS-345541) to study the role
70
of NF- B in CD40 protection. PDTC has been shown to inhibit NF- B activation (Wang et al.,
2008) and our results also show that PDTC prevented completely CD40-induced activation of NF-
B (IV, Fig.2B). Specific IKK-inhibitor has been shown to inhibit both IKK and IKK (Burke et
al., 2003). In two of the FL cell lines in which CD40 stimulation protected from TRAIL- and
cytotoxic drug-induced apoptosis, inhibition of NF- B prevented the protection indicating that
CD40 induced protection is completely dependent on NF- B (IV, Fig.2A, 3A). This was shown
only with TRAIL-induced apoptosis which is induced already in 4 h because inhibition of NF- B
pathway became toxic itself at the later time points as the number of apoptotic cells started to
increase slowly after four hours (data not shown). In addition, we show that CD40 could not protect
from NF- B-inhibitor induced apoptosis confirming that CD40 signaling that leads to protection
from apoptosis requires NF- B (IV, Fig.3B). In contrast, it was recently shown that CD40 ligation
mediated inhibition of NF- B and promoted apoptosis in human Burkitt lymphoma cell lines
(Wang et al., 2008). In HF4.9 cells, CD40 might also induce apoptosis by inhibition of NF- B but
further studies are still required to prove that hypothesis.
Inhibition of NF- B induced apoptosis in all three FL cell lines (IV, Fig.3B, 5B) indicating that
there is basal level of NF- B-regulated anti-apoptotic proteins in a cell which is not enough to
prevent TRAIL- or cytotoxic drug-induced apoptosis but is enough to protect from spontaneous
apoptosis. Furthermore, our results showed that inhibition of NF- B further sensitized cells to
apoptosis (IV, Fig.2A, 3A). It has also been previously shown that inhibition of IKK sensitizes
mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level (Roue et al., 2007). In
addition, it has been shown that CD40 induces up-regulation of c-FLIP and Bcl-XL and that the
induction is partially prevented by selective NF- B inhibitor, BAY 117085 (Travert et al., 2008).
Our RT-PCR results showed that CD40 induced expression of IAP family of proteins (c-IAP1, c-
IAP-2, XIAP, survivin), Bcl-XL and c-FLIPs (c-FLIP-L, c-FLIP-S) and the induction was
completely prevented by NF- B inhibition (IV, Fig.2C, 3C). At the protein level, CD40 induction
of these anti-apoptotic proteins was much less prominent. However, inhibition of NF- B decreased
considerably amounts of Bcl-XL and FLIP-S/L proteins (basal level) (IV, Fig.3D).
The anti-apoptotic proteins, FLIP-S and FLIP-L, block death receptor induced apoptosis inhibiting
activation of caspase-8 at the death inducing signaling complex (DISC) (Krueger et al., 2001;
Scaffidi et al., 1999). Overexpression of either of the FLIP isoforms inhibited TRAIL-induced
apoptosis but as supposed not intrinsic apoptosis induced by dexamethasone or doxorubicin (IV,
Fig.4). Follicular lymphomas overexpress Bcl-2 protein but it is not itself enough to prevent
71
TRAIL-, doxorubicin- or dexamethasone-induced apoptosis. Here we show that simultaneous
overexpression of Bcl-XL prevented almost completely dexamethasone- and doxorubicin-induced
apoptosis (IV, Fig.4B). In addition, Bcl-XL prevented TRAIL-induced apoptosis in mitochondria
dependent type II FL cells (HF28RA) but as supposed inhibited only partially TRAIL-induced
apoptosis in mitochondria independent type I FL cells (HF1A3) (discussed previously, III, Fig.1 ).
72
6. CONCLUSIONS
Glucocorticoids such as dexamethasone are frequently used in the treatment of lymphoid
malignancies due to their ability to induce apoptosis in lymphoid cells. However, the detailed
mechanisms and regulation of Dex-induced apoptosis remain still largely unknown in FL cells.
Dex-induced apoptosis depends on mitochondria since overexpression of Bcl-XL completely
prevented Dex-induced apoptosis. Protein synthesis is required for Dex-induced apoptosis already
before mitochondrial changes. The kinetics of Dex-induced upregulation of Bim correlates with
kinetics of apoptosis indicating that newly synthetized Bim might be the central mediator of Dex-
induced apoptosis. However, sh-RNA knockdown of Bim is required to prove the result.
Interestingly, activity of GSK-3 is required for Dex-induced upregulation of Bim and apoptosis.
Surprisingly, caspase-9 which is normally thought to be activated downstream of mitochondria,
regulates Dex-induced mitochondrial changes since overexpression of dominant negative caspase-9
prevented Dex-induced mitochondrial changes and apoptosis. (Fig. 18)
Figure 18. Mechanisms and regulation of Dex-induced apoptosis.
73
The inhibition of constitutively activated PI3-kinase-Akt pathway in FL cells enhances and
accelerates Dex-induced apoptosis indicating the importance of this pathway in the protection of
follicular lymphoma cells from cell death. Therefore modulation of PI3-kinase-Akt pathway
activity could provide new strategies to improve current therapeutic regimens in FL. Specific
inhibitors of PI3-kinase-Akt pathway should be considered in the future in the treatments of
lymphoid malignancies in combination with glucocorticoids. In addition, shifting the balance of the
activation of the kinases towards the state where glycogen synthase kinase-3 is kept active favors
an apoptotic response and could potentiate Dex-induced apoptosis.
TRAIL has gained lately a lot of attention because of its ability to induce apoptosis in cancer cells
leaving the normal cells intact. TRAIL induces apoptosis either by type I or type II pathway in FL
cells (Fig.19). Interestingly, in the presence of PDTC, TRAIL induced the full activation of
caspase-3 and apoptosis independently of mitochondria in Bcl-XL overexpressing type II cells
indicating that apoptotic pathway switches from type II to type I. However, the target of PDTC
remains to be solved. The activation of apoptosis independently of mitochondria could have great
advantage to treatment of FL because most cases of FL overexpress anti-apoptotic mitochondrial
Bcl-2 protein which either protects or delays apoptosis.
Figure 19. The possible mechanisms of type I and type II apoptotic pathways in FL cells in
response to TRAIL.
74
Furthermore, our results suggest that microenvironmental CD40 signals might have an impact on
treatment of FL. Interestingly, CD40 stimulation either protected from TRAIL- and drug-induced
apoptosis or induced apoptosis itself, depending on the maturation stage of FL B cells. Therefore,
the use of blocking CD40 antibodies in the treatment might not bring advantage for all FL patients.
Instead, since CD40 protection was dependent on NF- B and inhibition of NF- B induced
apoptosis in all three FL cell lines, the best combination for FL therapy according to our in vitro
studies could be pharmacological inhibitors of NF- B combined with TRAIL or other cytotoxic
drugs. Although inhibitors of NF- increasingly are being used for treatment of human
malignancies, serious side effects limit their clinical use. Thus, a preferable approach would be to
block the critical downstream anti-apoptotic targets of NF- rather than NF- itself.
75
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