CD137 ligand induced apoptosis in peripheral blood mononuclear cells (PBMCs)

i
CD137-INDUCED CELL DEATH IN PERIPHERAL
BLOOD MONONUCLEAR CELLS
NURULHUDA BINTE MUSTAFA
(B.Sc(Hons.), NUS)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF
SCIENCE
DEPARTMENT OF PHYSIOLOGY, MEDICINE
NATIONAL UNIVERSITY OF SINGAPORE
2010
ii
ACKNOWLEDGEMENTS
Oh humble graduate student how thy toils make thee weary!
Be not for the Grace of God and thy teachers, family and friends, thou wouldst have
surely perish.
To Professor Shazib Pervaiz for the rigorous development of a scientific mind
through your intellectual contributions and critical insights, for challenging me to rise
to the occasion, for your understanding and patience, for nurturing an amazing lab
which prizes friendship and love as much as Science, it will be difficult to find a
better atmosphere to mature in, a profound Thank You.
To Associate Professor Herbert Schwarz for always being there to assist and propel
me in my research, for the unique broadening of my scientific perspective which
comes when working with a co-supervisor in a different field of research and for your
quiet encouragement, you have been an invaluable pillar of support in my progress
and I am most grateful.
To Dr. Jayshree, Ms. Kartini, Dr. Andrea and Dr. Alan, Thank You very much for your
guidance especially at the initial stages as a naive UROPS student and throughout the
course of my work, for the endless cups of coffee over science, enjoyable chats and
selfless assistance beyond research.
To each and every member of the ROS and Tumour Biology lab and the CD137

Immunotherapy Lab whom I have worked closely with, you guys know who you are.
We brave not only the trials of scientific progress together, but also really care for
each other’s personal life, you guys are like family. Special mention to the class of
2005, Inthrani, Sinong, Chew Hooi, Zhi Xiong and Greg for inspiring each other to
continue marching down this difficult road and Shaqireen for being my sparring
partner in CD137 research.
To mom and dad, you have been unbelievable. For motivating me to always strive
for excellence, for your precious prayers, unflagging love and support, and most
importantly for your steadfast faith in me, words of gratitude are completely
inadequate for all you have given me. To my siblings Adibah, Nabil and Hazi, thank
you for inspiring me in ways that you do not know.
Finally to my husband, you alone truly know the rainbow of emotions and challenges
that I have experienced in the course of my scientific research and in writing this
thesis. For being willing to make any sacrifice so that I can focus on and pursue my
graduate studies, , for painstakingly putting pieces together when they fall apart and
for your dedication towards my best well being, you are my nikmah, my miraculous
blessing.
The pursuit of knowledge at its core is a commitment to apply new discoveries for
the transformation and betterment of the society. May this be the beginning of such
an endeavour.
iii
TABLE OF CONTENTS
I. Acknowledgements ii
II. Summary ix
III. List of Figures xi
IV. List of Abbreviations xiv
1.0 Introduction 1
1.1 Cell Death 1
1.1.1 Overview of Cell Death 1
1.1.2 Apoptotic Cell Death 1

1.1.3 Molecular Mechanisms Mediating Apoptosis 3
1.1.4 Extrinsic, Intrinsic And Granzyme B Cell Death Pathways 4
1.1.5 Caspase-Independent Cell Death 8
1.2 Apoptosis In the Immune System 10
1.2.1 Activated Induced Cell Death in T cells (AICD) 11
1.2.2 Activated Cell Autonomous Death in T cells (ACAD) 12
1.2.3 Monocyte-Dependent Cell Death in T cells (MDCD) 13
1.3 CD137/CD137 Ligand, TNFR/TNF Superfamily Members 15
1.3.1 Molecular Characteristics and Expression Patterns of CD137 17
1.3.2 Effects of CD137 Signalling 18
1.3.2.1 CD137 Signalling in T cells 18
1.3.2.2 CD137 Signalling in B cells 19
iv
1.3.2.3 CD137 Signalling in Dendritic Cells 20
1.3.2.4 CD137 Signalling in Granulocytes and Natural Killer Cells 20
1.3.3 Clinical Significance of CD137 21
1.3.4 Molecular characteristics and expression pattern of CD137 Ligand 23
1.3.5 Effects of CD137 Ligand Signalling 24
1.3.5.1 CD137L signalling in Monocytes/Macrophages 24
1.3.5.2 CD137L signalling in Dendritic Cells 26
1.3.5.3 CD137L signalling in B cells 27
1.3.5.4 CD137/CD137L mediated inhibitory signalling 27
1.4 Reactive Oxygen Species 31
1.4.1 Major Types of ROS and their derivative species 32
1.4.2 Superoxide Anion 32
1.4.3 The family of NADPH Oxidases 33
1.4.4 Hydrogen Peroxide 38
1.4.5 Hydrogen Peroxide-Mediated Cell Death 39
1.4.6 Oxidative Stress and Endogenous Antioxidant Defence Mechanisms 41
1.4.7 Redox Signalling in Peripheral T cells 43

1.4.7.1 ROS-mediated T cell Proliferation 43
1.4.7.2 ROS-mediated T cell Death 44
1.4.7.3 Control of the Extrinsic Apoptotic Pathway by ROS-dependent
Expression of FasL 44
1.4.7.4 Control of the Intrinsic Apoptotic Pathway by the
ROS-dependent Suppression of Bcl-2 Expression 45
v
1.4.7.5 ROS-mediated effects on T cell fate by Accessory Immune Cells 49
1.5 Aim Of Study 50
2.0 Materials and Methods 51
2.1 Chemical Reagents 51
2.2 Antibodies for Immunoblotting 52
2.3 Fluorescent dyes used for Flow Cytometry 52
2.4 Induction of CD137L signalling in Target Cells 53
2.5 Cells and Cell Culture 54
2.5.1 Isolation of Peripheral Blood Mononuclear Cells 54
2.5.2 Isolation of Mice splenocytes 55
2.5.3 SGH-MM6 cell line 56
2.6 Assays for proliferation and apoptosis 56
2.6.1 MTT Proliferation Assay 56
2.6.2 Morphological and quantitative analysis of cell size 57
2.6.3 Analysis of Phosphatidyl Serine Externalisation 58
2.6.4 Determination of Mitochondrial Transmembrane Potential 58
2.6.5 Analysis of Caspase Activity 59
2.7 Determination of protein expression levels by Immunoblotting 60
2.8 Mitochondrial and Cytosolic Isolation by differential centrifugation 62
2.9 Nuclear and cytosolic extraction………………………………………… ……………………………62
2.10 p65NF-κB Transcriptional (DNA Binding) Assay 63
2.11 TNF Enzyme –Linked Immunosorbent Assay (ELISA) 64
2.12 Assays for the Determination of Reactive Oxygen Species 64

2.12.1 Analysis of intracellular hydrogen peroxide production 64
vi
2.12.2 Analysis of intracellular superoxide anion production 65
2.12.3 Analysis of mitochondrial superoxide production 65
2.12.4 Cell specific depletion of ROS in a mixed T cell and
Monocyte reaction 66
2.13 Statistics………………………………………………………………………………… ………………….66
3.0 Results………………………………………………………………………………… ……….………………….67
3.1 Induction of CD137L signalling by immobilised CD137-Fc inhibits
proliferation and stimulates apoptosis in three different cell types 67
3.1.1 CD137L signalling inhibits proliferation of activated human PBMCs 67
3.1.2 CD137L signalling also inhibits proliferation in activated mouse
splenocytes and in multiple myeloma cell line, SGH-MM6 68
3.1.3 CD137L mediated decrease in cell proliferation is due to
induction of apoptosis 74
3.2 CD137L induced cell death is caspase independent and is
mediated by the intrinsic not the extrinsic death pathway 78
3.2.1 CD137L signalling increases expression levels of death receptor TNFR1 and
stimulates secretion of TNF, but is not critical to cell death 78
3.2.2 Inhibiting TRAIL-mediated signals with DR4 and DR5 blocking
antibodies was unable to abrogate CD137-induced cell death 82
3.2.3 Ligation by CD137-Fc disrupts the mitochondrial transmembrane
potential, and induces translocation of Cytochrome C into the cytosol 84
3.2.4 CD137L downregulates Bcl-2 protein levels at early time points,
while maintaining Bim levels thus tilting the Bcl-2: Bim
ratio towards favouring apoptosis 87
vii
3.2.5 CD137L signalling induces slight caspase-3 and-8 activity
at 24h but cell death is caspase independent 89
3.2.6 CD137-induced cell death is independent of casein kinase 1

(CK1) but is dependent on the MAPK pathway 92
3.3 CD137 induces apoptosis specifically within the CD3
+
T cell
subpopulation of the PBMCs and T cell death is mediated by monocytes 95
3.4 Cell death induced by CD137 is critically regulated by
Reactive Oxygen Species (ROS) 100
3.4.1 CD137-induced cell death can be abrogated by scavengers
of ROS, Catalase and Tiron 100
3.4.2 CD137 induces significant production of ROS in whole PBMCs 104
3.4.3 CD137L signalling induces ROS in both T cells and monocytes 107
3.5 It is ROS from T cells and not monocytes that is critical for the
induction of CD137-mediated cell death 113
3.6 The source of ROS in T cells that activates the cell death program
could be the NADPH Oxidase (NOX) or the mitochondria 115
3.6.1 Production of ROS critical to apoptosis is upstream of the
disruption in mitochondrial transmembrane potential 115
3.6.2 ROS in T cells originates from NOX 115
3.6.3 Inhibiting superoxide dismutases (SOD) with DDC
sensitised PBMCs to cell death 122
3.6.4 ROS in T cells may originate from mitochondria 128
4.0 Discussion 131
viii
4.1 CD137L-induced cell death is mediated via a caspase-independent
intrinsic (mitochondrial-mediated) pathway 132
4.2 CD137L mediated cell death signalling in PBMCs is targeted to T cells 137
4.3 CD137/CD137L induced cell death is critically mediated by
Reactive Oxygen Species (ROS) 141
4.3.1 ROS is the upstream molecular event crucial for stimulating the
CD137-induced death signalling cascade leading to T cell apoptosis 142

4.3.2 ROS potentially mediates CD137-induced T cell apoptosis
by downregulating the expression of Bcl-2 via the activity of ERK 143
4.4 Critical ‘killer’ ROS is produced by T cells not monocytes, though
cell death is monocyte dependent 146
4.5 ROS species that is ultimately responsible for the induction of cell death
signalling is H
2
O
2
which is produced from the activity of NOX 149
4.6 So how is CD137/CD137L induced cell death physiologically relevant? 153
4.6.1 CD137/CD137L: The missing link in monocyte regulated T cell death? 154
4.7 Summary and Conclusion 159
5.0 References 164
6.0 Appendix 185
6.1 Supplementary Figures 185
6.2 Publication and Poster 192
ix
SUMMARY
CD137-INDUCED CELL DEATH IN PERIPHERAL BLOOD MONONUCLEAR CELLS
Nurulhuda Mustafa
National University of Singapore, 2010
CD137/CD137L are members of the TNFR/TNF superfamily that have exhibited significant
immuno-modulatory effects in healthy and pathogenic states. Anti-CD137 antibodies have
shown great promise in murine therapeutic models of cancer and autoimmunity. Generally,
CD137/CD137L bi-directional signal transduction greatly amplifies the ongoing immune
response as CD137 signalling is co-stimulatory for T-cells while CD137 Ligand (CD137L)
signalling is activating for antigen presenting cells (APCs).
In spite of the solid evidence for CD137-induced survival signalling in T-cells, it was found
when CD137 was knocked out of splenocytes (1-3), splenic T cells responded not by hypo-

proliferation, but by hyper-proliferating instead (4). We set out to clarify this paradox and
identified that when immobilised CD137-Fc cross-links its corresponding ligand (CD137L) on
PBMCs, it activates an inhibitory signal which results in a reduced proliferative response to
anti-CD3. This potentially explains why deficiency in CD137 would conversely allow cells to
hyper-proliferate. We further determined that the lack of proliferative response is actually a
function of cell death in PBMCs and that apoptosis is targeted to T cells. T cell death peaks at
24h, and there is a dose-dependent increase in apoptosis with increasing concentrations of
CD137-Fc added to the PBMC culture. We found contrary to expectation that although there
is a CD137-mediated upregulation of death receptors, CD137-induced apoptosis is not
promoted by the extrinsic pathway but by the intrinsic pathway. We found a CD137-
x
mediated downregulation of Bcl-2 levels, while exerting no effect on Bim levels thus
favouring an increase in the Bim: Bcl-2 ratio which subsequently leads to mitochondrial
membrane permeabilization, the release of Cytochrome C and cell death. Despite the
translocation of Cytochrome C into the cytosol, we observed only low levels of caspase 9 and
caspase 3 activity and confirmed with a pan-caspase inhibitor that indeed that cell death is
caspase- independent.
We discovered instead that induction of cell death is critically dependent on reactive oxygen
species (ROS) production. We report that ROS production is not a secondary effect of
mitochondrial permeabilization but an early event initiating a death signalling cascade. ROS
production was stimulated as early as 2h which returns to baseline at 6h but subsequently
rises stably from 12h till 24h. We confirm that the key ROS species involved in cell death is
hydrogen peroxide (H
2
O
2
) and that H
2
O
2

production is NADPH oxidase (NOX)-dependent.
Thus, superoxide produced by NOX is potentially converted to H
2
O
2
by superoxide
dismutases in the cell.
Directly ligating purified T cells with CD137-Fc does not induce cell death. Concomitant
studies demonstrated that CD137-mediated cell death is induced only in the presence of
monocytes and increases with increasing monocyte concentrations. CD137-signalling
actually induces increase in ROS levels in both monocytes and T cells and by specifically
depleting ROS from T cells or monocytes we ascertained that the ROS which activates T cell
in PBMCs is produced by T cells themselves.
Altogether our data show that CD137 regulates a mechanism for caspase-independent T cell
apoptosis which is mediated by the intrinsic pathway and critically regulated by H
2
O
2
. Since
cell death is requires the presence of CD137-activated monocytes, we propose a novel
physiological role for CD137 in mediating monocyte dependent T- cell death (MDCD).
xi
LIST OF FIGURES
Introduction
Figure A: Three pathways leading to activation of caspases, the central executor of
apoptosis
Figure B: Expression Profile of CD137 and CD137L
Figure C: NADPH Oxidase (NOX) Family Members and Regulatory Subunits
Figure D: Reactive Oxygen Species (ROS) regulates T cell Death by modulating
expression of FasL and Bcl-2

Results
Figure 1: CD137L signalling inhibits proliferation of PBMCs, mouse Splenocytes and
multiple myeloma cells SGH-MM6.
Figure 2: CD137L induces decrease in cell size of PBMCs, a morphological feature of
apoptosis.
Figure 3: CD137L induces phosphatidyl serine (PS) externalisation in PBMCs.
Figure 4: CD137L upregulates TNFR-1 expression in PBMCs.
Figure 5: CD137L signalling significantly elevates cytokine TNF secretion from PBMCs.
Figure 6: Blocking the signalling through DR4 and DR5 was unable to reverse CD137L-
induced cell death.
Figure 7: CD137L signalling stimulates mitochondrial depolarisation and
hyperpolarisation.
Figure 8: CD137L enhances expression of Cytochrome C and induces translocation of
Cytochrome C from the mitochondria into the cytosol.
Figure 9: CD137L signalling in PBMCs suppresses the expression of Bcl-2 while having
no effect on Bim expression.
Figure 10: CD137L signalling does not significantly activate caspase 9 and only slightly
activates caspase 8.
Figure 11: CD137L mediates slight increase in caspase 3 activity which corroborates
with slight PARP cleavage
xii
Figure 12: Pan caspase-inhibitor ZVAD-FMK is unable to prevent CD137L-mediated
apoptosis.
Figure 13: ERK inhibitor PD98059 but not Casein Kinase 1 inhibitor IC261 can rescue
CD137L-mediated cell death in PBMCs.
Figure 14: CD3
+
T cells, and not CD3
-
cells purified from CD137L-treated PBMCs were

targeted for apoptosis.
Figure 15: CD137L targets cell death induction to the CD3
+
subpopulation of cells in
PBMCs.
Figure 16: CD137L-signalling through purified T cells only induces a marginal amount
of apoptosis as compared to PBMCs. However, T cell death is increasingly sensitised
in T cell-monocyte co-cultures as the concentration of monocytes increases.
Figure 17: Catalase was able to significantly reduce CD137L-mediated apoptosis.
Figure 18: Tiron completely abrogated CD137L-induced apoptosis.
Figure 19: CD137L signalling increases the population of PBMCs that contain
elevated H
2
O
2
levels.
Figure 20: CD137L mediates a temporal increase in H
2
O
2
levels which precedes cell
death at 24h.
Figure 21: CD137L signalling directly through purified T cells does not produce H
2
O
2
in T cells.
Figure 22: CD137L induces H
2
O

2
production in both the CD3
+
and CD3
-
subpopulation
of cells in PBMCs.
Figure 23: Increasing the concentration of monocytes to T cells decreased instead of
increased ROS production.
Figure 24: The population of CD3
+
T cells, in the 1:1 T cell: monocyte cell culture,
which contain low levels of ROS are dead/dying cells.
Figure 25: It is the ROS produced by T cells and not monocytes that is critical to
CD137-mediated T-cell apoptosis.
Figure 26: Production of ROS critical to CD137L-induced apoptosis is upstream of
mitochondrial depolarisation as Tiron is able to inhibit CD137-mediated loss in
transmembrane potential.
Figure 27: Inhibiting the activity of NOX can inhibit CD137L-mediated apoptosis.
Figure 28: CD137L signalling induces increase in expression of NOX subunits gp91
phox
and rac1 in T cells.
xiii
Figure 29: H
2
O
2
production mediated by CD137L signalling can be blocked by
catalase, superoxide scavenger Tiron and NOX inhibitor DPI.
Figure 30: There is a CD137L-mediated boost in superoxide production preceding the

vast amount of H
2
O
2
production at 24h.
Figure 31: Inhibitor of superoxide dismutase (SOD), DDC sensitizes PBMCs to CD137-
induced cell death.
Figure 32: DDC is able to reverse CD137L-mediated H
2
O
2
production but amplified
CD137L-mediated superoxide production.
Figure 33: CD137L signalling does not mediate any change in MnSOD and CuZnSOD
expression levels in T cells.
Figure 34: Ligation of CD137L on PBMCs leads to the increase in percentage of cells
with elevated mitochondrial ROS production.
Figure 35: Mitochondrial ROS production increases in the presence of monocytes.
Discussion
Figure 36: Proposed Model of CD137-activated monocyte dependent apoptosis in
PBMCs.
Appendices
Supplementary Figure 1: CD137L induced phosphatidyl serine (PS) externalisation in
multiple myeloma cell line SGH-MM6.
Supplementary Figure 2: CD137L signalling significantly elevates cytokine TNF
secretion from SGH-MM6 cells.
Supplementary Figure 3: CD137L-induced apoptosis in SGH-MM6 cells are TNFR-
independent.
Supplementary Figure 4: CD137L induces translocation of p65NF-κB from the cytosol
into the nucleus in PBMCs.

Supplementary Figure 5: CD137 induces activation of p65 NF-κB DNA binding activity
in PBMCs.
Supplementary Figure 6: CD137L induced phosphatidyl serine (PS) externalisation in
both OKT3 activated and non activated PBMCs.
Supplementary Figure 7: There is a dose dependent increase in cell death induced in
PBMCs with increasing concentrations of recombinant CD137-Fc.
xiv
LIST OF ABBREVIATIONS
AIDS Acquired Immunodeficiency Syndrome
AIF Apoptosis Inducing Factor
Apaf-1 Apoptotic Protease-Activating Factor-1
APC Antigen presenting cell
AML Acute Myeloid Leukemia
ATP 2-adenosine 5’-triphosphate
Bad Bcl-2 Antagonist of cell Death
Bak Bcl-2 Antagonist/Killer
Bax Bcl-2 Associated X protein
Bcl-2 B-cell Lymphoma protein 2
BH Bcl-2 homology
Bid BH3 Interacting Domain Death Agonist
Bim Bcl-2 Interacting Mediator
C-terminus Carboxy Terminus
CAD Caspase Activated DNase
CARD Caspase Recruitment Domain
Caspase Cysteine-dependent aspartate-specific protease
CED Caenorhabditis elegans death genes
CD137 Cluster of Differentiation 137
CD137L Cluster of Differentiation 137 Ligand
xv
CD3 Cluster of Differentiation 3

CHO Chinese Hamster Ovary
CK1 Casein Kinase 1
Cu/Zn SOD Copper/zinc superoxide dismutase
Cyt. c Cytochrome c
DC Dendritic Cell
Duox Dual Oxidase
DCFDA 5-(and-6)-chloromethyl-2',7'dichlorodihydrofluorescein
diacetate acetyl ester dichlorofluorescein diacetate
DD Death domain
DDC Diethyldithiocarbamate
DED Death effector domain
DEVD-AFC N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-
trifluoromethyl coumarin
DISC Death-inducing signaling complex
DMSO Dimethyl sulfoxide
DPI Diphenyliodonium
DR Death receptor
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
EGTA Ethyleneglycotetraacetic acid
ELISA Enzyme Linked Immunosorbent Assay
xvi
EndoG Endonuclease G
ER Endoplasmic reticulum
ERK Extracellular regulated kinase
ETC Electron transport chain
FACS Fluorescence activated cell sorter
FADD Fas-associated death domain-containing protein
FBS Fetal bovine serum
Fc region Fragment/crystallizable region

FITC Fluorescein isothiocyanate
fmk Fluoromethylketone
GSH Glutathione
GSSG Oxidised Glutathione
H
2
O
2
Hydrogen peroxide
Hepes 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
hr Hour
DHE Di-hydroethidine
IAP Inhibitor of apoptosis protein
IETD-AFC N-Acetyl-Ile-Glu-Thr-Asp-7-amino-4-
trifluoromethyl coumarin
Ig Immunoglobulin
JNK c-Jun N-terminal kinase
kDa kilodalton
xvii
KLH keyhole limpet hemocyanin
LEHD-AFC N-Acetyl-Leu-Glu-His-Asp-7-amino-4-
trifluoromethyl coumarin
MAPK Mitogen activated protein kinase
MAPKK Mitogen activated protein kinase kinase
MEK Meiosis-specific serine/threonine protein kinase
MDTCD Monocyte Dependent T cell Death
Mitosox
TM
Hexyl triphenylphosphonium cation (TPP
+

)-HE
MnSOD Manganese superoxide dismutase
mM milimolar
MOMP Mitochondrial outer membrane permeabilization
mRNA Messenger RNA
MTT 3-[4,5-dimethyl-2-hiazolyl]-2,5-diphenyl
tetrazolium bromide
NADPH Nicotinamide Adenine Dinucleotide phosphate
NFкB Nuclear factor of kappa light polypeptide gene
enhancer in B cells
NOX NADPH Oxidase
NP-40 Nonidet P40
N-terminus amino terminus
O
2
•
ˉ Superoxide radical
OH
•
Hydroxyl radical
xviii
PAGE polyacrylamide gel electrophoresis
PARP Poly(ADP-ribose) polymerase
PBS Phosphate buffered saline
PE Phycoerythrin
Phox Phagocytic oxidase
PI Propidium iodide
PI3K Phosphatidylinositol-3-kinase
PKC Protein kinase C
PMA Phorbol 12-myristate 13-acetate

PMSF Phenylmethylsulfonyl fluoride
PUMA p53-upregulated mediator of apoptosis
RIP Receptor interacting protein
RNA Ribonucleic acid
RNase Ribonuclease
ROS Reactive oxygen species
RPMI 1640 Rosewell Park Memorial Institute 1640
RTK Receptor tyrosine kinase
SDS Sodium dodecyl sulphate
SDS-PAGE SDS-polyacrylamide gel electrophoresis
SEB staphylococcal enterotoxin B
Ser Serine
siRNA small interfering RNA
xix
SLE systemic lupus erythematosus
SMAC Second mitochondrial activator of caspases
SOD Superoxide dismutase
tBid Truncated Bid
TCR T cell Receptor
TEMED N,N,N’,N’-tetramethlethylenediamine
Thr Threonine
TNF Tumor necrosis factor
TNFR Tumor Necrosis Factor Receptor
TRAF TNF receptor associated factor
TRAIL TNF-related apoptosis inducing factor
Tyr Tyrosine
VDAC Voltage dependent anion channel
XIAP X-linked inhibitor of apoptosis protein
zVAD-fmk Benzyoxycarbonyl valanyl alanyl apartyl-fluromethyl
ketone

1
1.0 INTRODUCTION
1.1 Cell Death
1.1.1 Overview of cell death
Cell death is a basic physiological mechanism utilised by organisms to remove
unwanted or excessive cells during growth and development, and to maintain tissue
homeostasis. Historically, cell death mechanisms have been classified as either a
regulated or an unregulated process. Regulated cell death or what is commonly
defined as apoptosis, refers to a cell’s resolution to commit suicide in response to
certain signals, by activating specialized death-inducing intrinsic cellular machinery.
Unregulated cell death, or necrosis, on the other hand occurs due to the
bombardment of overwhelming physico-chemical stresses on the cell which
inevitably leads to the cell’s demise. However, emerging evidence suggests that the
classic dichotomy between apoptosis and necrosis is an over-simplification of a
highly sophisticated mechanism. Recent studies indicate that there are several non-
apoptotic regulated cell death mechanisms that can occur concurrently with or
independently from apoptosis such as necroptosis and autophagy.
1.1.2 Apoptotic Cell Death
Apoptosis was the first form of regulated cell death to be discovered in 1972 (6) and
was characterised in Caenorhabditis elegans (C.elegans) in the early 1990s (7).
Briefly, the transcriptional upregulation of pro-apoptotic protein Bcl-2 homology 3
(BH3)-only protein, egl-1, sequesters the anti-apoptotic C.elegans death genes (CED)-
2
9, which relieves the inhibition CED-9 exerts on CED-4. CED-4 is now free to activate
cysteine protease CED-3, thereby allowing the cleavage of specific cellular substrates
which lead to induction of cell death (8).
Extensive genetic studies of apoptosis in mammalian cells have revealed that the
apoptotic mechanism in mammals is very similar to that of C.elegans, except that it is
much more complex. For every class of CED proteins, there exist multiple
mammalian homologues. The highly conserved and uniform nature of apoptosis

suggests that it is a tightly controlled process and a critical regulatory mechanism for
the normal development of living organisms. Indeed, the dysregulation of apoptosis
can lead to various disease pathologies. The uncontrollable proliferation of cells due
to a suppression of cellular apoptotic mechanisms can give rise to cancer. Apoptotic
protease activating factor 1 (Apaf-1)-deficient mice which have evidently lower levels
of apoptosis show significant abnormalities in the development of the brain (9). On
the other hand, excessive activation of apoptosis have been identified as the primary
cause of tissue injury and functional decline in a great number of acute diseases such
as stroke and chronic diseases such as diabetes (10) Due to its integral role in
maintaining tissue homeostasis, it is important for scientists to be able to correctly
identify cells undergoing apoptosis. Currently, it is generally accepted that it is best
to distinguish apoptosis morphologically, and that morphological features of
apoptosis are characterised by the rounding up of cells, reduction in cellular volume,
chromatin condensation, plasma membrane blebbing and phosphatidyl-serine
externalisation (11).
3
1.1.3 Molecular Mechanisms Mediating Apoptosis
Central to the execution of apoptosis are the cysteine proteases known as caspases
(cysteine aspartic acid specific proteases). Caspases are highly specific proteases that
cleave protein substrates containing particular tetra- or penta-peptide recognition
sequences at the aspartic residues (12). The proteolytic activity of caspases is
targeted to literally hundreds of proteins, amongst the most important is the
cleavage of major structural features of the cell such as actin, tubulins and nuclear
lamins which results in the rounding up of cells, cell shrinkage, membrane blebbing
and nuclear fragmentation (13-14). Due to its destructive effects, caspases are
typically present in healthy cells as inactive precursor enzymes known as zymogens
(15). Caspase activation is induced by a processing event that rearranges the
proteolysed large and small sub-units into a heterodimer which allows the catalytic
site to organised into the active conformation (15). There are currently twelve
caspases that have been identified in mammalian cells. Caspases are classified as

either initiator or effector caspases. Upon oligomerization and self-activation,
initiator caspases such as caspase 8 ,9 and 10 mediate the cleavage and activation of
inactive pro-forms of effector caspases such as caspases 3,6,7 which are responsible
for the ultimate disassembly of the cell (15).
At each stage of caspase activation, there is an additional level of regulation
provided by caspase inhibitors such as Flice-inhibitory protein (FLIP) and c-IAP
(inhibitor of apoptosis protein) which can directly inhibit caspase 8 and caspase 3
respectively (16-17).
4
It is worthy to note however that mammalian caspases are not only involved in
apoptosis. Caspase 1 for example, is activated during an innate immune response
and regulates inflammatory cytokine processing by cleaving IL-1β and IL-18 into
mature peptides (18) while deeper elucidation of the role of caspase 8 has
highlighted its role in contributing to T-cell proliferation (19).
1.1.4 Extrinsic (Death Receptor), Intrinsic (Mitochondrial) and Granzyme B Cell
Death Pathways
It is commonly accepted that there are three main pathways that lead to the
ultimate activation of caspases during apoptosis (Fig A). In the extrinsic pathway,
pro-apoptotic signals are delivered via DISC (death-inducing signalling complex)
which is assembled due to oligomerization of transmembrane death receptors (DR)
belonging to the TNFR superfamily such as Fas, TNFR1 (Tumour necrosis factor
receptor 1), DR4, DR5 , upon binding by their cognate ligands (20). The trimerization
of transmembrane death receptors stimulates the recruitment of adaptor proteins
such as Fas associated death domain protein (FADD) which in turn recruits and
facilitates the dimerization of caspase 8, thereby promoting its auto- activation.
Active caspase 8 can then process caspase 3 and 7 into mature forms that will trigger
further substrate proteolysis culminating in the termination of the cell (21).
Extrinsic death signals can cross-talk with the intrinsic death pathway via the caspase
mediated cleavage of BID (BH3-interacting domain death agonist). Like other
members of the BH3-only family, truncated BID is able to lead to the release of the

5
Figure A: Three pathways leading to activation of caspases, the central executor of
apoptosis
The extrinsic pathway is activated by oligomerization of death receptors which
form the death inducing signalling complex (DISC) activating caspases 8 which
cleaves and activates effector caspases 3, 6, 7.
The intrinsic pathway is activated by stimuli that can be detected by BH-3 only
proteins, thereby sequestering anti-apoptotic Bcl-2 proteins or directly
activating of Bax so that it can oligomerize on the mitochondrial membrane
and facilitate release of Cytochrome C into the cytosol where it can complex
with caspases 9, apaf-1 and ATP to form the apoptosome which stimulates
caspases 9 to cleave effector caspases.
Granzyme B is released into the target cells through the plasma membrane via
perforin and cleaves BID or effector caspases to initiate cell death.
Adapted from Taylor, 2007.
6
mitochondrial Cytochrome C which promotes the formation of the large apoptosome
complex consisting of Apaf-1 and caspase 9. Apoptosome formation leads to the
stimulation of caspase 9 activity and subsequent processing of effector caspases 3
and 7 (22).
In contrast, the intrinsic cell death pathway is regulated by the balance of the pro-
and anti- apoptotic members of the Bcl-2 family and by mitochondria derived
molecules such as hydrogen peroxide and pro-apoptogenic factors such as
Cytochrome C. Within the Bcl-2 family of proteins exist three subfamilies
represented by archetype molecules: (i) Bcl-2-like molecules that are anti-apoptotic
and contain all or most of the Bcl-2 homology (BH) domains 1–4; (ii) Bax-like
molecules that are pro-apoptotic and contain BH domains 1–3; and (iii) ‘‘BH3-only’’
molecules that are pro-apoptotic and whose only homology to Bcl-2 is in their BH3
domain. This latter group is the largest, consisting of members that are expressed in
a tissue-specific manner and appear to function upstream of the Bax-like molecules

(23-24). The initiation of the intrinsic cell death pathway can be mediated by a
variety of stimuli (such as drugs or reactive oxygen species) that provoke cell stress
or damage resulting in the activation of one or more members of the pro-apoptotic
BH-3 only protein family. For example, DNA damage induced by etoposide or 5-
Flurouracil can lead to a p53-dependent transcriptional upregulation of PUMA (25).
On the other hand, cell survival mediated the epidermal growth factor receptor
involves a mechanism which suppresses the intrinsic apoptotic pathway by
phosphorylating and inactivating BAD (26).