GENERATION OF OXlDATlVE STRESS BY
THE RESPIRATORY CHAIN FOLLOWING TREATMENT
WlTH DNA DAMAGING AGENTS
by
Nhu-An Pham
A thesis submitted in confomity with the requirements
for the degree of Master of Science
Graduate Department of Medical Biophysics
University of Toronto
O Copyright by Nhu-An Pham, 1999
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GENERATION OF OXlDATlVE STRESS BY
THE RESPIRATORY CHAIN FOLLOWING TREATMENT
WITH DNA DAMAGING AGENTS
Nhu-An Pham, Master of Science, 1999
Department of Medical Biophysics
University of Toronto
ABSTRACT
The generation of mitochondrial reactive oxygen intermediates (ROI) and expression of the
antioxidants glutathione and thioredoxin were examined in OCIfAML-2 cells following treatment
with the DNA damaging agents cytosine arabinoside, etoposide and y-radiation. A flow cytometry
method was developed for the assessment of rnitochondrial ROI and membrane potential in
digitonin pemeabilized cells under various respiratory conditions.
Advantages of this new
method are: (1) mitochondrial ROI and respiratory chain activity can be assessed at the single
cell level; (2)
ROI generation by non-mitochondrial sources are excluded.
Cells were
pemeabilized and assessed for respiratory chain activity 24 hr post-treatment to the cytotoxic
agents. Treated cells mat were still capable of generating a rnitochondrial membrane potential
with the respiratory substrates (malate/glutarnate, succinate), were obsenred to have higher ROI
generation than untreated, control celfs. Associateci with the oxidative stress is the increased
expression of glutathione and aiioredoxin.
Thioredoxin regulates redox senstive protein
sulphydryls that are associated with potentiating cell survivaf.
Thetefore, mitochondrial ROI
generation appears be involved with the induction of protecüve mechanisms, in addition to
mediating cytotoxicity,
1 would like b express my gratitude to my supervisor, Dr. David Hedley,
for his enthusiastic support and guidance.
Thank you for the unforgelable
experience of being on the "Stamhip Enterprise."
Sincere thanks to Dr. B&n Robinson and fie members of his laboratory
for insights into the wonders of the mitochondtial world. Many thanks to the
members of the Hedley laboratos, especially Sue Chow and Vojislav Vukovic. 1
would also like to acknowledge Dr. lona Weir for her support in the end moments
of cornpiethg this thesis, and
fo Dr, Roberf Bmce whose encouragement haû
started thisjoumey.
To my parents, whose love and support have made this possible. Thank
you.
Table of Contents
Abstract. ...............................................................................................ii..
Acknowledgements.............................................................................. ..III
Table of Contents..................................................................................
iv
List of Tables........................................................................................
vii..
List of Figures....................................................................................... vii
C hapter 1:
INTRODUCTION
1.1
ACUTE MYELOID LEUKEMIA................................................... 2
1.2
DRUG TREATMENT OF AML.................................................... 3
12 1
Cytosine Arabinoside....................................................... 3
1.2.2
TopoisomeraseIllnhibitors................................................ 4
1 .2.3
Induction of Apoptosis by Chemotherapy.............................. 6
1.3
CELLULAR RESISTANCE TO DRUG TREATMENT...................... 6
1.4
APOPTOSIS..........................................................................7
14 1
The Apoptotic Cascade..................................................... 7
1.4.2
Release of Cytachmme c and Cornmitment to Cell Death........-12
1.4.3
Regulation of the PemeabiIÏty 1ransition Pore....................... 14
REACTIVE OXYGEN INTERMEDIATES...................................... 17
1-5
1.5.1
1.5.2
Cellular Sources of ROI.................................................... -17
Sources of ROI in Apoptosis.............................................. -19
THE MITOCHONRIAC RESPIRATORY CHAIN............................. 21
1.6
16 1
Energy Production...........................................................
1.6.2
Mitochondriaf ROI Generation............................................ 24
ANTlOXlDANT SYSTEMS........................................................
1 -7
21
25
1.7.1
Defense Systems Against ROI........................................... 25
1 .7.2
Defense Systems Against Protein Thiol Oxidation.................. 25
THIOREDOXIN.......................................................................
1 -8
27
1.8.1
Regulatory Roles of Thioredoxin......................................... 27
1 .8.2
Thioredoxin in Cancer...................................................... -28
1.8.3
Thioredoxin and Oxidative Stress............. ,-.
1.9.1
ROI, Thioredoxin and Apoptosis. ........................................ 30
1.9.2
ThesisOutline................................................................
.................... 28
THESIS RATIONAL AND OUTLINE............................................ 30
1.9
1.10
REFERENCES......................................................................
31
33
THE DEVELOPMENT OF A FLOW CWCWETRY METHOD TO EXAMINE
MITOCHONORIM RESPIRATORY CHAIN ACTIVITY AND ROI GENERATION
IN DIGITONIN PERMEABIUZEDCELLS
Chapter 2:
2.1
ABSTRACT............................................................................ 41
2.2
INTROOUCTION..................................................................... 42
2.3
MATERIALS AND METHODS................................................... 43
Reagents....................................................................... 43
Cell Culture. ................................................................... -44
Respiratory Buffer............................................................ 44
Digitonin Permeabilizationof Cells....................................... 45
Assay Conditions for Mitochondfial RespiratoryChain
Activity..........................................................................-45
Respiration Measurements................................................
46
ATP Measurements..........................................................
47
Confocal Microscopy Analysis of Plasma Membrane
Integrity, ROI and A Y m.....................................................
-47
Flow Cytometry Analysis of ROI and AYm........................... -48
Statistics........................................................................ 49
2.4
RESULTS..............................................................................
49
2.4.1
Digitanin Perrneabilizationof Cells...................................... -49
2.4.2
Respiration Measurements................................................. 50
2.4.3
ATP Measurements......................................................... -53
2.4.4
Detection of Plasma Membrane Perrneabilization. ROI
And A T m by Confocal Microscopy....................................... 54
2.4.5
Analysis of Mitochondria1 ROI and A m
In Perrneabilued Cells by Flow Cytometry............................ 57
2.5
DISCUSSION......................................................................... 59
2.6
REFERENCES.......................................................................
62
Chapter 3: INVOLVEMENT OF MITOCHONDRIA1ROI GENERATION. AND
THlOREDOXlN IN APOPTOSIS INDUCED BY DNA DAMAGING AGENTS
3.1 ABSTRACT..............................................................................
65
3.2 INTRODUCTION..................................................................... A36
3.3 MATERIALS AND METHODS...................................................... 68
3.3.1 . Reagents......................................................................... 68
3.3.2. Cell Cufhrre....................................................................... 69
3.3.3. Dmg and Radiation Treatment ............................................ -69
3.3.4.
Flow Cytornetry Anaylsis of Oxidative Stress,AYm
and Plasma Membrane lntegrity ........................................... 69
3.3.5. Thioredoxin Measurements... .............................................. -71
3.4 RESULTS................................................................................. 72
3.4.1
Dnig toxicity Characterized by Loss of Plasma Membrane
Integrity and AYm.. ........................................................
3.4.2
Involvement of Oxidative Stress in Early and Late Phases
Of Apoptosis. .................................................................
3.4.3
73
Assessrnent of Mitochondnal ROI and AYm Dunng Dnig and
Radiation Treatment........................................................
3.4.4
-72
76
Thioredoxin Expression During Apoptosis............................. 81
3.5
DISCUSSION...........................................................................
3.6
REFERENCES.......................................................................... 91
85
CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS
4.1 SUMMARY.......................................................................................
96
4.2 FUTURE WORK................................................................................
99
4.3 REFERENCES.................................................................................
-102
Table 1-1
Effectors of the Permeability Transition Pore........................... 14
Table 1-2
States of Respiratory Control................................................ 23
CHAPTER 1
Figure 1-1
Structure and Metabolic Pathways of AraX .............................4
Figure 1-2
Structures of Daunorubicand Etoposide ................................ 5
Model for Apoptosis... ......................................................... 9
Figure 1-3
Figure 1 4
Figure 1-5
PenneabilityTransition Pore................................................ 13
Redox Sensitive Vicinal Sulphydryls of the Andenine
Nucleotide Translocator...................................................... -16
Figure 1-6
Supemxide Generation by Daunorubicin... .............................. 17
Figure 1-7
Reactive Oxygen f nterrnediates.............................................
Figure 1-8
Schematic Overview of M i h o n d n a l ROI Generation
in Apoptosis.....................................................................
18
-20
Figure 1-9
Oxidative Phosphorylationin Mitochondna.............................. 21
Figure 1-10
Mitochondrial Respiratory Chain.......................................... - 2 2
Figure 1-11
Inhibitors of the Respiratory Chain.........................................
Figure 1-12
Interaction of Antioxidant Systems......................................... 26
Figure 1-13
Mechanism of Redox Regulation in Bacteria............................ 29
24
CHAPTER 2
Figure 2-1
Time Course For Digitonin Permeabilkation of Cells.................. 50
Figure 2-2
Measurement of Equilibrated Oxygen Levels in Liquid Phase......51
Figure 2-3
Respiration in Whole and Digitonin Perrneabilized Cells............. 52
Figure 2-4
ATP Standard Curve........................................................... 53
Figure 2-5
ATP Production in Cells.......................................................
Figure 2-6
Efïects of Digitonin Permeabilizationon
54
Plasma Membrane lntegrity and ROI Labeling
Analyzed by Confocal Microscopy......................................... 55
Figure 2-7
Effects of Digitonin Permeabilizationon A Y m and ROI
Analyzed by Confocal Microscopy......................................... 56
Figure 2-8
Flow Cytomeûy Analysis of Respiratory Chain Activity with
Succinate in Digitonin Pemeabilized Cells.............................-57
Figure 2-9
Respiratory Chain Acüvii wiai Various Efbctors... ...................58
CHAPTER 3
Figure 3-1
Cytotoxic Effecb of DNA Damaging Agent.............................. 73
Figure 3-2
Oxidative Stress Following Dmg and Radiation Treatment-........ -74
Figure 3-3
Oxidative Stress in Early Apoptotic Phase. ............................. -75
Figure 3 4
Effects on Mitochondrial ROI and AYmFollowing
Ara-C treatment. ................................................................ 77
Figure 3-5
Mitochondrial ROI Gerteration in OCIfAML-2
24 hr Post-Treatrnent.........................................................
Figure 3-6
78
Mitochondrial Membrane Potential in OCIIAML-2
24 hr Post-Treatment with................................................... -79
Figure 3-8
Effects of Ceramide on Respiratory Chain Activity.....................80
T i t i o n Curve For the Monoclonal Antibody
Against Thioredoxin... .........................................................82
Figure 3-9
Changes in Thioredoxin Expression with
Figure 3-7
Etoposide Treatment ......................................................... 83
Figure 3-10
lncreased Thioredoxin Expression with Oxidative Stress
Associateci treatrnents in OCI/AML-2...................................... 84
CHAPTER 1
INTRODUCTION
1.'1 ACUTE MYELOID LEUKEMIA
Leukemia is cancer of the bone marrow and blood characteunwntrolled growth of blood cells.
by the
There are four major categories:
myelogenous or lymphocytic. which are further divided into acute or chronic.
Acute myeloid leukemia (AML) is the most common type of adult leukemia,
comprising about 40% of leukemias in the Western wodd (q). In most cases the
cause of AML is not evident, though risk factors associated with AML include
increasing age. patients with certain wngenital chromosomal abnomalities (e.g.
Down's syndrome. Fanconi's anemia), patients with myelodysplastic syndromes
and other pre-leukemic states, and environmental factors (exposure to ionizing
radiation, chernical toxins such as benzene, and treatment with alkylating agents)
(2. 3).
AM1 is characterized by failure of normal myefopoiesis. and the
accumulation of undifferentiated stem cells in the bone mamw and peripheral
blood. The stem cells are a heterogeneous population varying in degree of
development which can be described by histochemical features, and in most
cases classified into one of the seven sub-claçsifications of the FA6 (FrenchAmerican-British) system, designated M l to M7 (2).
Some FA6 types are
associated with cytogenetic abnormalities, such as chromosomal translocations,
or chromosomal deletions, or losses which have been associated with favourable
and unfavourable prognosis (43).
At present, a subtype-specific treatment
based on cytogenetic abnomalities is useâ for acute promyelocytic leukemia
(M3). where the differentiating agent, all-trans-retinoic acid produœs a high rate
of complete remission in patients. However, the role of cytogenetic in general
remains unclear in disease progression and in targeting of treatment modalities.
Chemotherapy only resuits in a small percentage of patients who are long terni
survivors.
Unfortunately, the majority of patients who achieve cornplete
remission using chemotherapy will relapse.
The median survival time is 12
months after relapse, with the leukemic cells becorning highly resistant to
conventional chernotherapy (6).
1.2 DRUG TREATMENT OF AML
12.1 Cytosine Arabinoside
The pyrimidine analogue cytosine arabinoside (ara-C) has been the
mainstay in the treatment of AML since its development in the early 1960s (7).
The metabolic pathways of ara-C are illustrated in Figure 1-1. It is taken up by
the nucleoside transporter and phosphorylated in cells to the active fom, ara-
CTP (cytosine arabinoside triphoshphate), or is rapidly dearninated to ara4
(uracil arabinoside) by the liver. The known mechanisms of ara-C cytotoxicity
are its inhibition of DNA polyrnerase and inhibition of DNA chain elongation and
chain termination (8, 9). Clinical treatrnent protocols for AML usually combine
ara-C with drugs that target DNA topoisomerase II.
Systemic
Cornoartmlent
.
h c
1
-
Target Ceil
Com~artrnent
-A~SC;)A~CMP
1,
Ara U
I
1
Ara UTP
I
I
ArâD CDP
i
VI
-
ArasTPI
r\
Cell
Membrane
CYTOSINE
ARABINOSIDE
'#
Ara UMP
dCTP
DNA
METABOLIC PATHWYS OF A R A 4
1.2.2 Topoisomerase Il Inhibitors
The
anthracycline,
daunorubicin
is
the
most
frequently
used
topoisomerase II inhibitor for the treatrnent of AML. Topoisomerase II is one of
the enzymes controlling the degree of supercoiling of DNA during DNA
replication and gene function. The proœss includes topoisomerase II binding to
the site of DNA distortion, leading to protein-associated double-strand breaks,
the passage of the strands to release the distortion, and the religation of the
broken strands. Anthracyclines increase protein-associated DNA fragmentation
by the inhibition of topoisomerase II religation of broken strands. and by
intercalation into DNA.
The structure of daunonibicin includes a planar
anthraquinone nucleus attached to an amino sugar (Figure 1-2). The quinone
moiety presents an additonal mechanism of toxicity that will be discussed in a
following
section. Interference with topoisomerase II acüvity is also mediated by
extracts from the mandrake plant (Podophyllum peltatum). A clinically effedive
derivative of podophyllotoxin is Etoposide (VP-16). The structure of etoposide
consists of a multiringed moiety, known as epipodophy/lotoxin, linked to the
sugar gluwpyranoside (Figure 1-2). Etoposide interferes Wh topoisomerase II
by forming cleavable enzyme-DNA complexes. resulong in single and double
DNA strand breaks (10, 11). Interestingly, these eady studies also observed that
though cells can rapidly reseal the DNA strand breaks once the dnig is removed,
cell death is not prevented (11, 12). This suggested the involvement of a death
process triggered by the DNA damage. Subsequent experiments identified the
process to be apoptosis (programmed cell death).
Figun 1-2. Strudures of anficanceragents daunwubicin, Mich is a gîycoside formed by
a tetracydic aglycone, with a quinone &iety, and an amino suga (Id),
and etoposide,
-
1.2.3 Induction of Apoptosis by Chemothenpy
The observation of extensive degradation of nuclear proteins and DNA
fragmentation occumng during death of kukemic cells treated with etoposide in
vitro suggests that chemotherapy induces apoptosis (13). A study by Gorczyca
et a/., dernonstrateci that apoptosis is also a significant death pathway in AML
patients undergoing chemotherapy. DNA fragmentation was found in leukernic
cells of peripheral blood during treatment with a r a 4 and e t o p i d e (14). The
therapeutic actions of numerous chemotherapeutic agents have now k e n
established by their in vitro apoptotic effect in different cell systems (1 5, 16, 17).
1.3 CELLULAR RESISTANCE TO DRUG TREATMENT
Drug resistanœ is the major obstacle in the treatment of cancer using
chemotherapy. Early studies identifid drug resistanœ mechanisms pnor to the
onset of DNA damage.
For example, dnig transport abnomalities (18, 19).
changes in target enzymes (20). or enzymes involved with drug metabolism (9,
21, 22), and with DNA repair processes (23).
In the treatrnent of AML with
daunorubicin. low expression of P-glywprotein, a membrane transport pump
associated with drug emux activq, favours induction of remission, though not the
probability of relapse (24). This suggests that multiple resistanœ mechanisms
are involved. Attempts to overcome drug resistana in AML have included the
use of high-dose ara-C, or the co-administration of an unrelated anticancer
agent. such as etoposide. These treatrnents show an increase in response rate
and response duration, but relapse still oawrs in the large majonty of patients
(6).
Failure to undergo apoptosis is a cornmon resistanœ mechanism that is
likely to explain a signifiant proportion of treatrnent failures. A plethora of recent
studies show that chemotherapy sensM.4iy of cancer cells is dependent on the
ability to trigger apoptosis (25-29).
It is now recognized that failure of dnig-
treated cells to undergo apoptosis-induced cell death is an important cause of
multidrug resistanœ.
1.4 APOPTOSIS
1.4.1 The Apoptotic Cascade
Apoptosis is a term proposed by Kerr, Wyllie and Cume in 1972 to
describe a wntrolled degradation process of the nucleus and cytoplasm
observed in cells during spontaneous cell death, or death triggered by
physiological or noxious stimuli (30). Cell death by apoptosis is distinct from
necrosis.
It is an active, regulated process involving biochemical and
rnorphological changes.
Studies
on
development of
the
nematode
Caenohabdith elegans identified the wre camponents of the muitistep proœss
(31). Essential gene products for apoptosis in C. elegans are CED-3, CED4,
and CED-9. Sinœ then, the mammalian homologues have k e n identified. The
genes CED-3 and CED4 promote apoptosis and are related to the mammalian
genes, known as caspases and apoptotic protease activating factors (Apafs).
Caspases are a family of cysteine proteases that cleave homoestatic and
structural proteins resulting in the typical manifestations of apoptosis, such as
plasma membrane and nuclear degradation. Caspases exist as zymogens until
activated by selfcleavage wïth cofactors. or by upstream, "initiatop caspases.
Apoptotic protease activating
factors (Apafs) are cofactors that a d to promote
proteolytic cleavage of caspases.
For example, Apaf-1 binds with cytosolic
cytochrome c (cyt c/Apaf-2) and deoxyadenosine triphosphate (dATP) to activate
pro-caspase 9 (Apaf-3) (32).
The gene CED-9 is related to the mammalian Bcl-2 gene family, which
includes two subgroups of proteins that eiaier inhibit or promote apoptosis by
targeting to the mitochondria, the endoplasmic reticulum and nuclear envelope
(33). Although the mechanisms of apoptosis regufation by the Bcl-2 gene family
are cunently unresolved, their effeds on regulation at the point of cyt c release
have been documented by many studies (33). The identification of the role of
such genes has since redirected apoptosis research away from the nucleus to
the cytoplasm. and most reœntfy to the mitochondria.
Many studies now suggest that the mitochondrial pathway of caspase
activation plays a central role in cell death induced by cytotoxic agents. An
overview of the present understanding of the apoptotic proœss is shown in
Figure 1-3. The apoptotic process is comprised of at least three functionally
distinct phases: induction, effector and degradation phase, with the mitochondria
having a key role (34-36). During the induction phase, a wide range of stimuli
can initiate the apoptosis proœss, such as DNA damage by chemotherapeutic
agents and radiation. or the activation of death receptors by tumour necrosis
factor u (TNFa) or Fas ligand. The signal transduction in the induction phase is
dependent on the stimulus and cell type. Mediators implicated in these 'private
pathwaysn include oxidative stress, ceramide, Ca*
and initiator caspases.
Oxidative stress and ceramide generation have been reported in apoptosis
triggered by TNFa, Fas ligand. a vanety of chernothefapeutic drugs and
radiation.
-
1O
extracellular
I
w
receptor mediated
signalling
1
initiator caspases --+
nucleus
effector caspases
1
-
primary necrosis
(without apoptosis)
apoptosis secondary necrosis necrosis
Figure 1-3. Model for apoptosis indudion
9
The generation of œramide is by activation of sphingomyelinase (3740),
a specific f o m of phospholipase C that hydrolyzes membrane sphingomyeiin to
generate the sphingolipid ceramide, or by the activation of ceramide synthase
(41).
It remains unclear as to the upstream mechanisms by which these
enzymes are activated during induction of apoptosis.
Support for the generation of reactive oxygen intemediates (ROI) as the
source of oxidative stress during apoptosis has k e n show at several ievels (42,
43). First, the addition of oxidants, or depletion of cellular antioxidants can resuit
in apoptosis. Second, apoptosis can be blocked by the addition or induction of
antioxidant enzymes, and thirdly, the generation of ROI has been associated with
induction of apoptosis.
The mitochondrial respiratory chain is a candidate
generator of ROI during apoptosis induced by chemotherapeutic agents as
supported by several independent groups. Previous work frorn our laboratory
has shown that there is an increase in cellular ROI early in ara4 and radiation
induœd-apoptosis (4445). Other groups have shown that ceramide, a mediator
involved with apoptosis by ara4 (39), acts to inhibit functioning of the
mitochondrial respiratory chain (46), thus increasing mitochondrial ROI
generation (47). One of the objectives of this study is to assess the significanœ
of mitochondria as a source of reactive oxygen intermediates (ROI) generation
during apoptosis triggered by araG and other DNA damaging agents such as
etoposide and radiation.
Oxidative stress and the other initiating events of apoptosis converge at
the effector phase where the mitochondria coordinate various induction signals
into common metabolic reactions.
Although. the mechanisms of signal
processing remain to be elucidated. the present knowledge recognizes that
changes in the mitochondrial metabolic events preœde nuclear apoptosis and
can be subjected to various regulatory mechanisms, such as by proteins of the
Bcl-2 gene family, and cellular redox regulators. such as the cellular antioxidants
glutathione and thioredoxin (42). The undedying hypothesis of this thesis is mat
folowing DNA damage, mitochondriaI ROI generathn msults in oxidative stress,
the consequenees of which can be boU, ha&/,
or pmtective due to the
induction of cellular antioxidant sysiems involving glutathione and thioredoxin.
The overall goal is to present a more cornplete understanding of oxidative stress
involvernent in the apoptotic process at the effector phase. where signals of cell
survival and cell death are k i n g integrated.
The outcorne of the effector phase is the release of apoptogenic factors,
such as cytochrome c (cyt c) from the mitochondria into the cytoplasm, the
induction of the mitochondrial penneability transition, characterized by the
dissipation of the mitochondrial membrane potential (AYm). and the loss of
cellular redox control. The release of cyt c initiates the degradation phase in
which "effector" caspases are activated
to bnng about the morphological
changes typical of apoptosis. These indude cytoplasmic shrinkage, membrane
blebbing. chromatin condensation and extensive DNA fragmentation.
The
cornmitment to cell death following cyt c release presented in the schernatic
shown in Figure 1-3 has two possible pathways. First, the rapid induction of
caspases resulting in apoptosis, and secondly by a slower necrotic cell death due
to metabolic consequences of mitachondrial damage.
1.4.2 Release of Cytochrome c and Commitrnent to Cell b a t h
Cytochrome c (cyt c) is nonally localized to the mitochondrial
interrnernbrane space, where it facilitates the transfer of electrons in the
mitochondrial respiratory chain.
It is released into the cytosol followïng the
induction of apoptosis by many difierant stimuli including cytokines (TNFa. Fas
ligand), chemotherapeutic agents and radiation (27, 32, 38. 48, 49).
Experimental observations suggest that the disruption of the mitochondrial
respiratory chain upon cyt c release could contribute to a caspase-independent
death mechanism that is associated with loss of ATP production and a state of
high oxïdative stress, driving the cell slowly towards a necrotic death (50-52).
In many apoptosis scenarios, the release of cyt c is observed to occur
simultaneously with the collapse of the mitochondriat membrane potential (AYm),
indicating the opening of a large conductance channel know as the mitochondrial
pemeability transition pore (PT pore). The penneability transition allows for the
passage of small proteins (1-5 KDa) into the cytosol(53). To date, the PT pore is
partially defined by the adenine nucleotide translocator (ANT), which is located
on the inner mitochondrial membrane, and the mitochondrial porin/voitage-
dependent anion channel (VDAC), located on the outer mitochondrial membrane.
These proteins are believed to moperate at sites where the inner and outer
mitochondrial membranes wme together to fomi a large conductance channef
(Figure 14).
CaUdeath m
r
s
:
oxidan6. Ca' ,
ceramide. aspases
Outer
MRodiondriai
Membrane
Figure 1 4 . A model showing some of the components of the permeability transition
pore. The open configuration allows the release of cyt c and oiher caspase-activating
proteins into the cytophsm. Cyclophilin and the peripheral ben#rdiiepine receptoc
are some of the proteins a3SOCialed with Vie regdation of the pore.
The opening of the mitochondrial pemeabifity transition pore (PT pore) facilitates
the release of cyt c by two possible mechanisms (34-36). In the first, the opening
of the PT pore, and wnsequently the entrance of water and solutes into the
matrix space causing osmotic disequilibrium, mitochondrial swelling and the
nipturing of the outer membrane. This resutts in the release of mitochondrial
proteins. including cyt c into the cytoplasm. The escape of cyt c in the second
model is by the opening of the outer mitochondrial membrane channel without
concomitant mitochondrial swelling. The second model may account for the late
occurrence of the colfapse of AY:, after cyt c release in some apoptotic systems.
In this case the penneability transition could be triggered by dysfunction of the
respiratory chain resulting from cyt c loss (53). The common outcome of boa
models is damage to the mitochondrial respiratory chain, a loss of redox wntrol,
the dissipation of AY,.
and the failure of mitochondrial ATP generation.
1.4.3 Regulation of the Pemeability Transition Pore
Table 1-1 Effectors of the Permeability Transition Pore
Function
Activator of PT
Inhibitor of PT
Thiol sensor
Prooxidants (menadione, te&
butylhydroperoxide), thiol-cross-linking
agents (diamide, phenylarsine oxide)
LOWAYm
Oxidation of NAD(PIH2
- . - -(in equilibrium with
GSH oxidation)
Alkalinization of matrix space pH (pH 7.3)
N-ethylmaleimide,
dithiolthreitol
Voltage sensor
Sensor of pyndine
- oxidation
Matrix space pH
sensor
- Cation sensor
ADPfATP sensor
1 Protease sensor
4
.
Ca*
1 Caspase 8
-
1
High AYm
Antioxidants (0hydroxybuty&e)
Neutral, or acidic matrix
Wace PH
Mg*, Zn*
ADP
1
The permeability transition pore (PT pore) is sensitive to a wide variety of
effecton (Table 1) (53, 54). Of interest to induction of apoptosis by
chemotherapeutic agents is the sensiavity of the permeability transition pore (PT
pore) towards the redox status of the cell. The induction of oxidative stress,
depending on cell type and apoptotic stimulus, has been associated with
generation of reactive oxygen intermediates (ROI) production, and modulation of
antioxidant defenses (42,43. 55).
Previous studies on the mechanism of ara-C toxicity in AML cells carried
out at the Ontario Cancer lnstitute impficate oxidative stress as a mediator of the
apoptotic death proœss. The antioxidant N-acetylcysteine has been shown to
protect against ara-C killing of OCIIAML-2 cells, in vitro (56). Furthemore, work
in our laboratory has shown eariy increases in ROI and glutathione levels, prior
to the loss of mitochondrial membrane potential in OCVAML-2 cells undergoing
apoptosis by ara-C and radiation.
Glutathione (GSH) is a ubiquitous and
abundant tripeptide thiol (y-glu-cys-gly). It scavenges free radicals in cells and
acts to maintain the cellular redox balance in a reducing state needed for cell
suivival. The increase in GSH during the early response to ara-C and radiation
is proposed as a protedive mechanism against the oxidative stress. Therefore,
ROI generation is important in triggering downstream events of the apoptotic
pathway occuring prior to the loss of AYm (44, 45). but it can also elicit a
protective response in AML cells. by the induction of antioxidants such as GSH.
Accumulating evidence suggests that oxidative damage to mitochondrial
sulphydryl (-SH) groups plays a critical role in the regulation of apoptosis. This
provides a biochemical mechanism that links the mitochondrial perrneability
translion pore to the overall redox regulation of the cell (57-62). Of particular
interest is the adenine nucleotide translocator (ANT), a component of the
penneability transition pore cornplex which has two redox sensitive vicinal thiols (SH)2on the inner side of the PT pore complex (Figure 1-5, (60)). Treatment of
cells with agents that are specific to thiol oxidation. resulting in the formation of
disulphide bonds (-S-S-), such as with diamide or phenyfarsine oxide (PhAs-OH),
induces the mitochondrial permeability transition more effedvely than with
prooxidants, such as t-butylhydroperoxide (t-BOOH). In accordance, disulphide
formation is prevented by reduction to dithiols with dithiothreitol (DTT), or thiol
substitution with Nsthylmaleimide (NEM) (5760, 62).
These experiments
suggest that sensitïvity to oxidative stress mediated apoptosis is dependent on
which the cumulative effects of ROI. the presenœ of antioxidant defense
systems scavenge ROI, and on systems that regulate reduction-oxidation states
of protein sulphydryls. The following Secfisecfions
present the cunent understanding
on these topics in the mechanism of apoptosis induction.
highlighting some
unresolved issues that will be addressed in this thesis.
-SH
DTT
-S
NEM
Figure 1-5. Effeds on vîtinal sulphyâryi gmps uf the adenine nudeotide transporter (AM).
Sulphydryl gmup oxidation by prooxidants (rnenadione, tBOOH, diafrîda), or fis cornplex
with PhAs-OH favours an open pore conformation. ReducGon MM
DTT,or subslitution with
NEM favoun a closed pan confoimation.
1.S REACTIVE OXYGEN INTERMEDIATES
1.5.1 Cellular Sources of ROI
The major reactive oxygen intermediates (ROI) in normal metabolic
processes are superoxide anion (027and the products of subsequent chernical
reactions of Oz'- which include hydrogen peroxide
radical (HO').
(H202),
and the hydroxyl
Sources of ROI include xenobiotics, cellular flavin enzymes such
as xanthine oxidase and NADPH oxidase, and the mitochondrial respiratory
chain.
Xenobiotics that participate in the production
of superoxide are typically
quinones, such as menadione. The quinone moiety undergoes a 1-electron
reduction to the corresponding semiquinone, a transitionary free radical which
then rapidly donates its extra electron to molecular oxygen, regenerating the
parent moiecule with the production of superoxide. Subsequent ROI generation
occurs via the pathways presented in Figure 1-7.
O
O
OH
Sugar
Figure 1-6. The 1-eledron redudion of t
kquinone moiety of dawrirbicin
to the reactim semiquinone ieads b the generationof superoxide as the
parent compoud is regenerated-
Because the
antifeukernicagent
dizunorubicin is a
quinone that is
capable of
generating ROI
(Figure I-6) (63),
experiments
described in
chapter 3 use the
alternative
topoisornerase II
inhibitor,
etoposide-
The enzyrnatic ROI generating systems indude the flavin enzymes which
use oxygen as a hydrogen acceptor in oxidation reactions resulting in the
generation of H202. For example, xanthine oxidase is involved in the catabolism
of purine bases to uric acid for excretion. NADPH oxidase consumes a large
amount of oxygen, known as the respiratory burst, which functions in phagocytic
cells to generate a rapid increase in HzOzdunng response to inflammation and
l
MITOCHONORIAL RESPIRATORY CHAIN.
RAVIN-DEPENDENT OXIDASE
I
WDRODGEN PEROXIDE
1
HABER-WEISS
REACTlON
1
HW+ OHHYDROXY RADICAL
Figure 1-7. Production of readive oxygen intemediates.
The major cellular source of ROI generation, however is from the normal
metabolic activities of the respiratory chain. It is calculatecl that approximately
1% to 4% of oxygen molecules reacüng with the respiratory chain are
inwrnpletely reduced to ROI in most cell types (64. 65). The estimated steadystate levels of liver mitochondrial generation of H202 and
are in the
nanomolar and picomolar range. respectively (66). A schematic outline of ROI
generation by flavin enzymes and the respiratory chain is presented in Figure 17. The reaction is initiated by the transfer of lelectron to molecular oxygen
yielding the superoxide anion, which rapidly dismutates spontaneously, or is
catalyzed by cytosolic or mitochondrial superoxide dismutases (CulZn SOD.
MnSOD) to give H20z. In the presenœ of transition metals. H202can then be
converted to the highly reactive HO' by the HaberWeiss reaction (42).
1S.2 Sources of ROI in Apoptosis
Sources of ROI production that have been implicated during apoptosis
include enzymatic systems and the mitochondrial respiratory chain. Examples
from the enzymatic systems include PIG3. an oxidoreductase enzyme originally
identified in a colon cancer cell line, whose expression is dependent on wild type
p53 activation (67).
Others include the NADPH oxidase (68) and xanthine
oxidase (69). However, the acüvation of these systems appears to be cell type
and apoptotic stimulus specific, and their inhibition does not prevent the ROI
increase under other apoptotic models (45, 70).
The mitochondrial respiratory chain has been implicated in the generation
of ROI under many different apoptotic stimuli of different cell types. Examples
include, hepatocytes (47, 71), heart cells (46), and in neuronal (70, 73),
fibrosarcoma (74, 75), and leukemic cell Iines (39, 75,76). Early ROI generation
during apoptosis has only been indirecüy linked to the rnitochondrial respiratory
chain. A putative mediator of a r a 4 induced-apoptosis, ceramide (39) has been
previously shown to inhibit the mitochondrial respiratory chah (45), and trigger
mitochondrial ROI generation in isolated mitochondria (47).
A schernatic
ovewiew of wolk that has implicated abbenant respiratory chain activity dunng
apoptosis induction is shown in Figure 1-8.
/ Apoptosis induction1
-----,
1
1 Oxidative Stress
?\"'
Permeability Transition,
release of cyt cl
cell death
Figure 1-8. Speculative involvement of mitochondrial reacüve oxygen intemediates as
mediators of apoptosis. Dashed lines indicate evidence from independent studies which
propose a mechanism between mitochondrial fundion and generation of ROI.
1.6 THE MITOCHONDRIAL RESPIRATORY CHAIN
1.61 Energy Production
The respiratory chah couples respiration to the generation of cellular
energy in the form of ATP, as presented in Figure 1-9.
1
lnner membrane
Outer membrane
-
Figure 1-9. The general mechanism of oxidative phosphorylation. Electrons are shuttled
along the respiratory chain and energy is released to pump H out of the matrix space
creating a membrane potenlia1 (&Y) and a proton gradient. The resufting electrochernicai
proton gradient across the inner membrane is used by the AT? synthase to generate ATP
from ADP and Pi in the rnatrk Facilitated exchange of rnatrix ATP with cytoplasmic ADP
is by the voltagdependent anion ctiannel (VDAC) and the adenine nucleotide translocator
(ANT). constituents of the pemeabiltiy transition pore (PT pore).
The mitochondrial respiratory chain consists of a series of redox-sensitive protein
complexes ernbedded in the mitochondrial inner membrane, and camers
(ubiquinone/Coenzyme Qlo and cytochrome c) that transfer elecrons between
these complexes (Figure 1-10).
-
Succinate
dehydrogenase
(Complex II)
NADH
L
NADH
dehydrogenase
(Cornplex 1)
1
Cytochrome b-c
(Complex III)
Cytochrorne oxidase
(Complex IV)
Figure 1-1O. The transfer of electrons (e O) through the respiratory main complexes
in the inner mitochondrial membrane. Ubiquinone (Q) and cytochrome c (c) are
mobile carriers that shuttle electrons from one complex to the next.
The protein complexes oxidize hydrogen derived from the oxidation of food
breakdown products (fatty acids, amino acids and carbohydrates) with rnolecular
oxygen to generate water at complex IV (cytochrome c oxidase/COX).
The
electrons carriad by NADH are transferred to respiratory complex I (NADH
dehydrogenase) and then to ubiquinone (Q). The electrons from succinate in the
trica rboxylic acid cycle are transferred to complex II (succinate dehydrogenase)
then to Q. Complex III then accepts the electrons from Q, and passes them to
cyt c. which shuttles them to aimplex IV where the final acceptor is % Oz to give
H20. The energy released by the electron flow is u s d by complexes 1, III and N
to pump protons (H3 ta the outside of the inner mitochondrial membrane, thus
creating an electrochemical potential (H'gradient and a AYm) that is positive on
the outside and negative on the mitochondrial matrix side. The transportation of
the protons back into the matrix by ATP synthase drives the synthesis of ATP by
the condensation of Pi and ADP.
ATP is then exported to the cytosof in
exchange for ADP by the adenine nucleotide translocator (ANT).
Chance and VVilliams have defined five conditions that can control the rate
of respiration in rnitochondria shown in Tabie 1-2 (77). These conditions control
respiration by the availability of ADP, reducing equivalents (NADH, succinate),
and oxygen. For example, state 4 would generally describe respiration of cells in
a resting state where the limiting factor is primarily ADP. For the purposes of this
study, the capacity of the respiratory chain is of interest. Thus state 3 is used.
where there are saturating amounts of ADP and reducing equivalents.
Table 1-2. States of Respiratory Control
State 1
State 2
State 3
State 4
State 5
Conditions Limiting the Rate of Respiration
Availabilitv of ADP and substrate
~Gilabilityof substrate only
The capacity of the respiratory chain itself, when al1 substrates
And components are present in saturating amounts
~vailabilityof ADP only
Availability of oxygen only
1.6.2 Mitochondrial ROI Generation
Potential ROI generation sites in the mitochondrial respiratory chain are at
the iron-sulphur components found at complex 1, II and III, where l-electron
reactions can occur with 02, rather than between the complexes and ubiquinone.
Experimental evidenœ shows that depending on the cell type, inhibition of
electron flow at complex I by rotenone (71). or complex III by antimycin A (47)
induces ROI generation (Figure1-11). tt is currently believed that this inhibition
prevents the transfer of electrons further along the respiratory chain, and
consequentiy their accumulation at the complexes before the site of inhibition
favoun their reaction with O2to give O*-.
S~eflate
NADH-
1-
1
FA0
Fes]
Q-
Rotenone
RFA
,-
Antimycin A
Corne x I l
Cyib,FeS,Cytc, c*Cytc-
Cornplex IV
r-i
Cyta3-02
w
Figure 1-1 1. Proposed sites of inhibition9 of the respiratory main by the chernical rotenone,
the antibiotic, antimycin A, and an Fe-ctielating agent, T F A . The eledron carriers include
complex I (oxidoreductase containing flavine rnononucleotide (FMN) and FeS); complex II
(oxidoredudase containing flavine adenine dinudeotide (FAD) and FeS); complexes Ill and IV
contain cytochrome oxidases.
1.7 ANTlOXlDANT SYSTEM
1.7.1 Defense Systems Against ROI
The involvement of reactive oxygen intemediates (ROI) during induction
of apoptosis suggests mat the cellular redox state, the balance between ROI and
antioxidant defense mechanisms. is central to the regulation of this process.
Cellular antioxidant systems can be functionally categorized into two groups: (1)
systems that scavenge free radical$; (2) systems that maintain protein
sulphydryls.
The primary enzymatic defenses against ROI include both the cytoplasmic
and mitochondrial forms of superoxide dismutase (CulZnSOD, MnSOD), which
convert superoxide to hydrogen peroxide (H202), and enzymes that detoxrfy
hydrogen peroxide. The major system involved in H202 detoxifjcation is the
glutathione redox cycle, which catalyzes the reduction of H20z by converting
reduced glutathione (GSH), to its oxidized f o m GSSG. The cycle is completed
with the regeneration of the substrate GSH by glutathione reductase using
NADPH as the donor of reducing equivalents.
1.7.2 Defense Systems Against Thiol Oxidation
Disulphide bonding (-SOS-)in proteins has major effects on conformation
and function.
Aithough the reducing environment inside cells favours
maintenance of free sulphydryl groups (-SH), the generation and redudion of
disulphides is now recognized as an active and specific process. The process of
apoptosis is an example in which the cellular redox state is a regdatory
mechanism. The sensitivity of the penneability transition pore (57-62), and of
other proteins involved in the apoptosis process. such as cytochrome c (80) and
caspases (81) towards sulphydryl redudion-oxidation states suggests that
disulfide bond formation rnay be a cornmon response mechanism to apoptotic
stimuli that involve induction
of oxidative stress.
Endogenous systems that maintain thiol redox control are glutaredoxin
and thioredoxin (82). Glutaredoxin and thioredoxin are smelt proteins of about
100 amino acid residues which have been characterized frorn a wide variety of
prokaryotic and eukaryotic species. As in the glutathione redox cycle. NADPH is
the source of reducing equivalents in the reduction of oxidized thioredoxin by
thioredoxin reductase. Glutaredoxin differs in that it may use either GSH or
thioredoxin in the regeneration of reduced glutaredoxin.
The interactions
between NADPH and the antioxidant systems of glutathione, thioredoxin and
glutaredoxin are illustrated in Figure 1-12.
I I IIVI
wuwnmm
reductase
-
nïoreaoxmJ
MT
/
/
S-S
I lI l O l G U U A l l l
1
T4TI S SH
Figure 1-12. Sumrnary of the intetadion of antirnidarit systems invohring glutathione. thioredoxin
and glutaredoxin. NADPH provide reducing equivalents to glutathione and thioredoxin.
Glutathione is the substrate for glutaredoxin reductcise in the mintemance of reduced glutaredoxin.
The systems of thioredoxin and gluataredoan both contain the same
redox-active disulphide site provided by the cysteine residues in the sequence,
Cys-Gly-Pro-Cys, which can donate hydrogens to Sglutathiolated proteins and
to protein disulphides.
Thioredoxin and glutaredoxin have greater reducing
potential and specificity toward proteins than is seen with GSH. Of interest to
this thesis is the regulation of thioredoxin during apoptotic induction, as it has
higher specificity towards the redudion
of disulphides than gluatredoxin which
favours more the reduction of S-glutathiolated proteins (83). Thus, thioredoxin
plays a major role in rnaintaining the redox States of the vicinal sulphydryls of the
pemeability transition pore, and of other apoptogenic proteins.
1.8 THIOREDOXIN
1.8.1 Regulatory Roles of Thioredoxin
Thioredoxin (TM) has several known biological functions (84). Orïginally,
it was identifid as a cofactor for ribonucleotide reductase, the fitst rate limiting
step in DNA synthesis. Its effect on cell growth is by several mechanisms, one of
which is its secretion by fibroblasts and cancer cells as a growth factor.
Thioredoxin can regufate other antioxidant systems, such as mediating the
uptake of L-cystine, a substrate for GSH synthesis (85), and by upregulating
MnSOD gene expression (86). It also exerts redox control by regulating the
activity of transcription factors, such as NF-KB which is responsive to oxidative
stress, and AP-1 and HIF-1 which are responsive to hypoxia (87)-
1.8.2 Thioredoxin in Cancer
Thioredoxin (TM) was fimt reported in hurnan T e l l lymphotrophic virus
1-positive T cells and natural killer cells. It was recognized as an inducer of
interleukin-2 receptoru, and thus termed ADF (aduit T-cell-derived factor (88).
Berggren et al., then observed that the expression of TRX mRNA and thioredoxin
reductase activity is elevated in human primary colorectal carcinomas in
cornparison to adjaœnt normal colonic mucosa, and mat both proteins have a
wide variability of gene expression in human hematologic and solid tumour cell
lines (89). Malignant hepatomas were observed to contain higher TRX protein
than normal liver tissue, and sensitivity towards the anticancer agent, cisplatin
was found to correlate with TRX mRNA levels (90). The transfdon of human
TRX cDNA into a number of different cancer cell Iines, including Jurkat,
fibrosarcoma, bfadder and prostatic cell lines resutted in increased resistanœ to
cytotoxic agents that produce oxidative stress, such as H202,cisplatin, mitomycin
Cl doxorubicin, etoposide, and UV irradiation (91-94).
1.8.3 Thioredoxin and Oxidative Stress
Thioredoxin (TM)is both induced by, and protects from oxidative stress.
The induction of TRX by oxidative stress has been demonstrated by several
groups of investigators. Work has k e n done using ischemia as the source of
oxidative stress (95, 96). For example, in cerebral ischemia due to carotid artery
occlusion, areas with the greatest neuronal death were associated with a time
dependent decrease in TRX mRNA.
Consistently, in adjaœnt areas where
neurons escaped death, TRX mRNA was obsecved to be upregulated (95).
lncreased T M mRNA has aQo k e n repo~edto occur due to other inducers
associated with oxidative stress induction, such as semm withdrawal and
hypoxia (89).
A potential mechanism for TRX feedback regulation is linked to its ability
to interact with stress adivated transcription factors such as NF-KB and AP-1
(87). This mechanism is exemplified by the baderial protein OxyR, illustrated in
Figure 1 1 3 (97).
Normal
Reducing
Conditions
Oxidative
Stress
Damaged
Reduce non-native
Diwlphides
Nemve
Dbulphide
Trigger
Degrade the Ondant
L
J
Figure 1-13. Cellular redox homeostasis in becteria provides a mode1for
redox regulation in mammalian systems. Oxidatiw stress oxidizes
sulphydryl groups to disulphide bonds leading to the inactivation of
cellular pfoteins and the activation of redox sensitive transcription
factors, such as OxyR. Active OxyR tfiggers the expression of enzymes
that degrade the oxidant and redua disulphide bonds. Reduction of the
disulphide bonds in OxyR provides for negative fesdback.
Disulphide formation on cellular proteins due to oxidative stress leads to
activation of OxyR (analogous to NF-KB), a transcription factor which regulates
the expression of enzymes that detoxify oxidants and reduce disulphide bonds.
The regeneration of protein disulphydryls by these antioxidant enzymes inhibits
OxyR activtty, thus providing negative feedback.
The mode1 presents an
attractive mechanism for aquired resistanœ towards
chernotherapedc dnigs mediated by oxidative stress.
the toxic effects of
The activation of the
stress responsive transcription factor NF-KB, associated with induction of genes
that function to prornote cell survival, has already been reported with in vfim
treatment of cells with a r a 4 (39) and etoposide (98), and a variety of other
cytotoxic agents (99, 100).
1.9 THESIS RATIONALE AND OUTLINE
1.9.1 ROI, Thioredoxin and Apoptoeis
Oxidative stress is involved dunng the induction of apoptosis in a wide
vanety of ceIl rnodels. However, the consequences are not well understood.
The generation of reactive oxygen intemediates (ROI), and systems to defend
cells against the oxidative stress have both been shown to be actively regulated
during apoptosis. The DNA damaging agents, a r a 4 and irradiation have been
shown by various groups of investigators to increase cellular ROI generation in
the early phase of apoptosis induction.
Mitochondna are implicated as the
generator of the ROI mediating the oxidative stress during apoptosis. However,
there is no direct evidenœ to demonstrate mitochondrial ROI involvement in
apoptosis induced by DNA damaging agents.
The interest in elucidation of
rnechanisms of ROI generation and the consequenœs of the resuiting oxidative
stress haî been initiated from the observations of the paradoxical roles
associated with ROI: a mediator of apoptosis, and an inducer of cell survival
mechanisms (e-g. antioxidants).
This thesis furthers the understanding of
oxidative stress induced by apoptotic stimuli (ara-C, etoposide and radiation).
The objectives are to assess involvement of mitochondrial ROI, and to assess
the regulation of the antioxidants, glutathione and thioredoxin following treatrnent
with cytotoxic chemotherapy and radiation.
1.9.2 Thesis Oudine
The temporal relationship between mitochondrial fundion and the
cytotoxic effects of DNA damaging agents cannot be elucidated entirely in the
intact ceIl. Aîthough previously reported studies have used extracts of isolated
mitochondrial particles to assay for mitochondrial alterations, this does not allow
for a direct appraisal of the behavior of those same mitochondria N, situ. Chapter
2 describes a novel method to assay for mitochondrial aiterations with respect to
ROI production and the maintenance of mitochondrial membrane potential using
fiow cytornetry. Using this method, data are presented in Chapter 3 to support
the hypothesis that during treatrnent of OCI/AML-2 cells with DNA damaging
agents (Ara-c, etoposide, radiation), the mitochondrial respiratory chain is altered
to increase production of ROI. Data are also s h o w that implicate this oxidative
stress in the upregulation of the antioxidants glutathione and thioredoxin.
Chapter 4 surnmarizes the experimental findings and discusses future
experimental directions.
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CHAPTER 2
THE DEVELOPMENT OF A FLOW CYTOMETRY METHOD TO
EXAMINE MITOCHONDRIAL RESPIRATORY CHAIN ACTlVlTY
AND ROI GENERATION IN DIGITONIN PERMEABILIZED CELLS
2.1 ABSTRACT
The mitochondrial respiratory chain is the major source of ROI generation
during normal metabolism. This chapter describes the developrnent of a novel
method to measure mitochondrial respiratory chain activity at the single œll ievel.
Cells suspended in a respiratory buffer were permeabilized by digitonin, labeled
with mitochondrial membrane potential (AYm) and ROI sensitive dyes. and
assessed for response to effectors of the mitochondrial respiratory chain using
flow cytometry. Other measures of respiratory chain acüvity. such as oxygen
consumption and ATP synthesis were also examined in the model. Addiaon of
ADP, and substrates for cornplex I (rnalate/glutamate), or complex II (succinate),
supported a lower ROI generation, and higher AYm, ATP synthesis and oxygen
consumption than cells in respiratory buffer with ADP alone.
Uncoupling the
respiratory chain from ATP synthesis with CCCP resulted in the dissipation of the
AT,, cessation of ATP production, and a higher generation of ROI. The use of
confocal microswpy direcüy demonstrated that intracellular ROI were fonned in
the mitochondria as shown by carboxy-DCF fluoresœnce. a probe for ROI,
localizing to energized mitochondria labeled with CMXRos. This technique using
digitonin peneabilized cells for the analysis of mitochondrial hrnction by fiow
cytometry allows the in situ assessrnent of mitochondrial ROI generation by
specific respiratory chah enzymes (e-g. complexes I and II). M i l e excluding
background signals from non-mitochondrial sources.
2.2 INTRODUCTION
The mitochondrial respiratory chain generates a significant amount of ROI
during oxidative phosphorylation (1). The respiratory chain consists of five multisubunit protein complexes embedded in the inner mitochondrial membrane
(Figure, 1-10), (2). Efedrons produced from the oxidation of nutrients are camed
by NADH or succinate and enter into the respiratory chain at camplex I or
cornplex II, respectively.
Ubiqoinone (Q, Coenzyme Q ~ o then
)
accepts the
electrons from either of these complexes, and passes them to wmplex III.
Complex III transfers the electrons to cytochrome c, which then shuttles them to
complex IV where the final acceptor is molecular oxygen to generate water. At
complexes 1, III and IV the energy released by the electrons is used to pump
protons
(H3
across the
mitochondrial inner membrane,
creating an
electrochemical gradient. The transportation of the protons back to the matrk
space is used to drive the production of ATP by ATP synthase.
Reactive oxygen intermediates (ROI) are produced when ubiquinone
intermediates from i4ectron reactions at the ubiquinone moiety transfer these
to molecular oxygen to generate superoxide, rather than mediating the transfer of
electrons between Q and complexes 1, II and III. Inhibitors of the respiratory
chain at these potential sites increase accumulation of the free radical
ubiquinone intemediate and exacerbate mitochondrial ROI generation.
For
example, rotenone, an inhibitor to complex Il and antimycin A, an inhibitor to
complex IIIcan potentiate ROI generation in different cell types (3-6).
Previous studies examining the role of the mitochondrial respiratory chain
in the generation of ROI during apoptosis have shown the followhg: 1) depletion
of mitochondrial respiratory enzymes confers resistanœ to apoptosis induction
(7, 8).
2) inhibitors of the respiratory chain enzymes potentiates apoptosis
induction by increasing mitodiondrial ROI generation (3-6);3) inhibition of
respiratory chain enzymes is observed early in apoptosis (4, 9-11).
These
observations suggest that the increase in mitodiondrial ROI generation is
mediated by damage to the respiratory chah
The method described in this chapter was developed to allow the
examination of mitochondrial ROI generation simultaneously with respiratory
chain function in digitonin pemeabilized cells, using flow cytometry. Presently,
there is no in situ assay available for the assessrnent of mitochondrial ROI
generation.
Data are presented showing that the digitonin permeabilization
system excludes background from non-mitochondrïal ROI generating sources.
Application of this method to study the temporal relationships betwean ROI
generation and respiratory chain activity during drug and radiation induœd
apoptosis is described in chapter 3.
2.3 MATERIALS AND METHODS
2.3.1 Reagents
Malate, glutamate, succinate, rotenone, antimycin A, carbonyl cyanide mchlorophenylhydrazone
(CCCP),
sucrose,
KH2PQ.
MgC12,
potassium
morpholinopropane sulphonate (MOPS), adenosine 5'diphosphate (ADP),
ethylenediaminetetraacetic acid (EDTA), essentially fatty acid free bovine serum
alburnin (BSA), propidium iodide (PI). and digitonin, were purchased from SigmaAldrich Canada Ltd. (Oakville, ON). The ATP determination kit, carboxy-DCFDA
(carboxydichlorofluorescin diacetate), chloromethyl-X-rosamine (CMXRos), and
DiICl(5) (.1,11,3,3,3',3'-hexamethylindodicarbocyanine) were
obtained from
Molecular Probes Inc. (Eugene, OR).
2.3.2 Cell CuRun,
OCVAML-2 cell line is a continuous Iine of acute myeloid leukemia blasts
originally isolated from a patient at Ontario Cancer lnstitutelPrincess Margaret
Hospital, obtained from Dr. McCulloch at the Ontario Cancer Institute (12). Cells
were grown in a-minimal essential medium (a-MEM), supplemented with 10%
fetal bovine serum (FBS), and maintained at 37OC. under a humidified
atmosphere of 95% air and 5% COz. The cells were grown at a concentration of
0 . 5 ~ 1 0to~2x10~cells/ml, and routinely subcultured every 2 3 days. Cells were
discarded every three months and re-established from frozen stock.
2.3.3 Respiratory Buffer
For experirnents, 1x10~cellslml were suspended in a respiratory buffer
wnsisting of 0.25 M sucrose, 2 mM KH2P04,5 mM MgCI2, 1 mM EDTA, 0.1%
fatty acid free BSA, 1 mM ADP, and 20 mM MOPS (pH 7.4), adapted from
previous studies using isolated mitochondria (3, 6, 10, 11, 13-15).
Sucrose,
KH2P04,and MgCI2 were us& for the osmotic support of the mitochondria in
digitonin pemieabilized cells. The bivalent cationic chelator EDTA was added to
bind contaminating ions, such as Ca* which can leak into the permeabilized
cells, or be released from intracellular stores (endoplasmic reticulum) when
treated with digitonin, and alter physiologie processes (15). The substrate ADP
was added to facilitate the coupling of respiration to ATP synthesis.
2.3.4 Digitonin Permeabilization of Celk
Selective digitonin pemieabilization of the plasma membrane was
originally used by Tager, et al., for the separation of mitochondria from the
cytoplasm of isolated rat-lier cells (14). Digitonin selectively damages plasma
membrane rather than the inner mitochondrial membrane as the cholesterol
content of the plasma membrane is much greater than that of the latter. Sinœ
digitonin reacts specifically with cholesterol, a concentration could be detemined
that penneabilires the plasma membrane H i l e leaving the mitochondrial
membranes intact. To allow entry of mitochondrial substrates and permit the
analysis of their effeds on the generation of mitochondrial ROI and mitochondrial
membrane potential, a concentration of digitonin which pemieabilkes the plasma
membrane of OCIIAML-2 while leaving the inner mitochondrial membrane intact
was detennined. Perrneabilïzation of the plasma membrane was detected by
entry of propidium iodide (PI) into cells as anafyzed by flow cytometry (see flow
cytometry set-up). Propidium iodide is a DNA dye which does not permeate the
intact plasma membrane of cells.
2.3.5 M a y Conditions for Miûxhondiial Respiratoy Chain Activity
5 mM malatel5 mM glutamate (maWglu) were used to generate
intramitochondrial NADH, which can donate a pair of electrons ta complex 1.
Electrons to cornplex II were provided by 5 mM succinate.
Inhibitors, 1OpM
rotenone, or 200 nM antimycin A were used to blodc electrons at complexes I
and III, respectively. Uncoupling of the respiratory chain from ATP synthesis was
achieved by the addition of 0.5 mM carbonyl cyanide m-chlorophenylhydrazone
(CCCP). This uncoupling agent dissipates the mitochondrial membrane potential
by acting as a proton ionophore across the mitochondrial inner membrane.
2.3.6
Respiration Measurements
Oxygen consumption by the mitochondrial respiratory chain was
monitored with a Clark type polarographic elecfiode. Yellow Springs Instrument
rnodel 53 (Yellow SprÏngs Instrument Co.. Inc.). A thin membrane was stretched
over the surface of the sensor in order to isolate it from the environment. VVhen
the polarizing voltage was applied across the membrane. oxygen reacted at the
cathode (O2 + 2H20 + 4e = 40H3 which caused a current to fiow through the
system. The amount of cunent that flowed was proportional to the amount of
oxygen that was exposed to the electrode.
The change in current output
reflected the amount of oxygen consumed by the system over time, and was
recorded on a graphic trace.
Oxygen consurnption was measured at a
concentration of 10x10~cells/ml in an assay volume of 3ml respiratory buffer at
37OC with constant stimng.
Calculations were made assuming air-saturated
respiratory buffer contains approximately 200 pM O2 (16).
consumption were expressed in nanograms/min/lx1os cells.
Rates of oxygen
2.3.7 ATP Measuremenîs
ATP synthesis was measured in digitonin pemeabilized cells after 30 min
of incubation at 37OC under the conditions assaying for mitochondrial respiratory
chain activity as described above. An aliquot was then taken and added to ice
cold deionized water, and kept frozen at -20°C until rneasurement of ATP by the
ATP determination kit (Mofecular Probes Cat. #A-6608). This kit is based on the
bioluminescence detection of ATP. using recombinant firefly luciferase and its
substrate luciferin. The reaction catalyred by luciferase requires ATP to produœ
Iight, as shown:
luciferin + ATP + Oz
""
-
oxyluciferin + AMP + PPi + COz + hv (560 nm)
Total chemiluminescenœ was collected by a Berthold Lumat LB9501
luminometer, and expressed as relative light units (RLU). The amount of ATP
frorn a test solution
was quantifieâ by cornparison to a calibration curve using
ATP as the standard.
2.3.8 Confocal Microscopy Analysis of Plasma Membrane Integrity, ROI
and AYm
The effeds of digitonin penneabilization on plasma membrane integnty,
mitochondrial ROI and A Y ~
were characterîzed by fluorescent probes and
visualized by confocal microswpy.
Whole cells, and pemeabilized cells
incubated with succinate were double labeled with 5 pgîml PI and 5 pM carboxyDCFDA. Whole cells and permeabilized cells were also doubled labeled with
5 pM carboxy-DCFDA and 90 nM chloromethyl-X-rosamine (CMXRos), a probe
for
A%
which is sequestered into the energued mitochondnal matrix spaœ
due to its cationic lipophilic structure. This was used as the probe for AYm
because the confocal microscope was not equipped with a redemiting laser
required for excitation of DiIC1(5), the probe used for flow cytometry. Cells were
incubated with the dyes at 37OC for 30 min, with the exception of PI which was
added ta the cells in the last 5 min. An aliquot of cell was then placed onta a
slide and observed using a Zeiss model LSM-510 inverted laser scanning
microscope equipped with a 488 nm air cooled argon laser. Propidium iodide
fluorescence and CMXRos fluorescence were collected with a 585 nm longpass
filter; carboxy-DCF fluoresœnœ was collected with a 525 nm 2 10 nm bandpass
filter.
2.3.9
Flow Cytometry Analysis of ROI and AY?,
Reacfive oxygen infennedrates.
Reactive oxygen intermediates (ROI)
were measured using 5 pM carboxy-DCFDA (carboxydichlorofluorescin
diacetate).
This probe passively diffuses into cells, where the acetates are
cleaved by intracellular esterases, fonning a negatively charged carboxy-DCF
product which results in retention of the molecule inside the cell.
A green
fluorescence results frorn the oxidation of carboxy-DCF.
Mitachondnal membrane potenfial. The mitochondrial membrane potential
(AT,)
was
detected by staining with 40 nM DilCl(5) (1,11,3,3,3',3'-
hexamethylindodicarbocyanine).
Being amphipathic and cationic, this probe
concentrates in an energized, negatively charged, mitochondrial m a t h space.
Cells were incubated with both probes at 37OC for 30 min following
perrneabilization with digitonin and treatment with the various conditions
assaying for respiratory chain adivity.
Flow cjdometry setup. The cells were analyzed using a Coulter Epics Elite
flow cytometer (Coulter, Florida). Two aircoolad lasers emitting Iight at 488 and
633 nm were used. For carboxy-DCF and PI excitation was at 488 nm and
fluorescence was measured using a 525 nm
+ 10 nm bandpass filter, and a 610
nm t I O nm bandpass filter. respectively. DilCl(5) was excited at 633 nm and
+
fluorescence was measured through a 675 nm 20nm bandpass filter.
2.3.1 0 Statistics
Experiments were repeated at least three times. Statistical analyses of al1
data were canied out using student's t-test, with statistical significanœ at p~0.05.
2.4 RESULTS
2.4.1 Digitonin Permeabilùption of Cells
The amount of digitonin required to pemieabilize cells at room
temperature was deterrnined by propidium iodide uptake, analyzed by flow
cytornetry over time. Figure 2-1 shows the time course for peneabilization of
the plasma membrane with 1 pg to 1O pg of digitonin per 1x10~
cells. Addition of
8 pg digitoninllxl~~
cells resulted in 100% permeability to PI after 4 min at r w m
temperature and was
therefore
chosen
for
subsequent
experiments.
Expenments were then done to establish that mitochondrial respiratory chah
activity (respiration, generation of ATP and AYm)was not compromised by these
penneabilization conditions.
O
1
2
IWE
3
4
5
m
Figure 2-1. Digitonin permeabilization as measured by entry of PI into
non-intact plasma membrane of cefls over tirne. Amount of digitonin
used is per 1x10' cells.
2.4.2 Respiration Measurements
Calibmtran of the oxygen probe. Figure 2-2 shows the calibration of the
oxygen probe to the respiratory bMer equilibrated to 21% (air). 6.26%, and 3.5%
02.
and N2gas at 37OC. Adjustrnent was made, setting the meter to read 0% in
N2 saturated respiratory buffet. A Iinear relationship was obserwd between the
equilibrated solution and the gases of various oxygen composition. Thus. meter
readings indicate the arnount of oxygen present in each rnilliliter of solution.
MEASUREMENTOF EQUlLlBRATED
OXYGEN LEVELS IN LlQUlb PHASE
O
5
10
1s
20
PERCENT Of OXYGEN IN GAS PHASE
Figure 2-2. Measurement of oxygen content in respiratory buffer
equilibrated with known amounts of oxygen gas at 37'C.
Respiration of OCVAML-2.
A typical oxygen consumption trace of
OCVAML-2 in respiratory bMer supplemented with ADP is shown in Figure 2-3.
After the intact cells reached a steady state of respiration. digitonin was added to
initiate pemeabilization.
Respiration decreased to a minimum immediately
following pemeabilization due to the diRusion of endogenous respiratory
substrates out of the cells. Addition of the complex I substrates malatelglutamate
to the pemieabilized cells re-induced respiration. Oxygen consumption œased
with the addition of the complex I inhibitor, rotenone, indicating that respiration
was substrate dependent
Succinate. the substrate for complex II was then
added and induced respiration at a rate sirnilar to whole cell respiration. Addition
of antimycin A inhibited the eiectron transfer at complex III, an acceptor of
electrons downstrearn of complex II, and thus inhibited oxygen consumption
induced by succinate at complex IV, where molecular oxygen is consumed in the
reaction with the ekctrons in the generation of water. Addition of CCCP to cells
being maintained by succinate resuhed in high levels of oxygen consumption due
to the uncoupling of oxidative phosphorylation. The ability to manipulate oxygen
wnsumption wîth the various respiratory chain Mectors confimis that the
respiratory chain is intact and functional m e n cells are suspended in the
respiratory buffer following pemieabilization with digitonin
Figure 2-3.A represmtative trace of oxygen consumption by whole and digitonin
permeabilized AML-2 cells. The addition of the various respiratory chain effectors
following pemeabilization were as follows: malatdglutatmate (mal/glu), rotenone
(ROT), succinate (SUC), antimycin A (AA), and uncoupler CCCP. The trace was
made with 10x10~ceils/ml in a total of 3 ml. Values are mean*SEM rates of
oxygen wnsumption in nanogram atoms par min par 106œllsfmm three separate
experiments.
2.4.3 ATP Measurements
ATP standard c u m An AT? standard curve was generated for each
experimental nin. Two readings were made for each known amount of ATP.
Figure 2 4 is a representative ATP standard curve showing
the relationship
between ATP concentration and relative light un& (RLU) collecteci using a
luminometer.
O
2
4
6
8
M
12
1
@roll
Figure 2-4. Relative IigM units (RLU) are measured for known anounts of ATP
and plotted as MEANf SEM dtwo difiarent sample readings.
ATP synthesis. The synthesis of ATP was detennined in the respiratory
buffer of digitonin penneabilized cells under vanous effectors of the mitochondrial
respiratory chain, as shown in Figure 2-5. ATP synthase activity requires an
electrochemical gradient and the substrates ADP and Pi for the synthesis of ATP.
Consistent with the biochemistry, ATP synthesis was not supported in the
absence of the respiratory chain substrates. or absence of ADP, or with the
addition of the uncoupler CCCP. The addition of ADP alone, or in combination
with the mitochondrial substrats significantly increaseâ ATP synthesis. The
small increase in ATP levels with the addition of ADP suggests that residual
endogenous respiratory substrates were present in the mitochondrial matrix
space to support ATP synthesis.
ATP PRODUCllON IN CELLS
74
,
0
MAU
GLU
WC
AûP
M A U SUC
GLU +AûP
+ ADP
CCCP
EFFECTORS
Figure 2-5. ATP production in AML-2 in respiratory buffer under selective conditions:
substrate for complex I (malatdglutamate) or complex il (succinate), with or without
ADP, and in the presenœ of the uncoupier CCCP. Values of ATP were calculated
from three separate experiments, mean S E M pmol per 10' cells incubated with the
effectors for 30 min at 37'C.
2.4.4 Detection of Plasma Membrane Penneabilization, ROI and A Y m by
Confocal Microscopy
Images were acquired of whole cells and digitonin permeabilized cells to
show the effects of digitonin treatrnent on plasma membrane integrity. and the
generation of mitochondrial ROI and membrane potential @Ym).
Plasma membrane integtfty and ROI. Figure 2 8 shows the images of
whole cells and permeabilized cells stained with PI and carboxy-DCF. The intact
plasma membrane of whoie cells was impermeable to the DNA fluorochrome PI
(A, Figure 2-6). The staining of PI in the nuclei of cells was observed following
disruption of the plasma membrane by digitonin (Cl
Figure 2-6). lntracellular ROI
was detected in both whole cells and penneabilud cells (B and D, Figure 28).
However, permeabilued cells showed the ROI labeling localizing to the
mitochondria (Figure 2-7).
WHOLE CELLS
DIGITONIN PERMEABILIZEDCELLS
Figure 2-6, Fluoresence images of OCVAML-2 cells labeled for plasma
membrane integrity (PI) and ROI (carboxy-DCF)- W o l e cells (A and B),
and digitonin perrneabilized cells with the addition of succinate (C and D)
were incubated witti carboxy-DCF far 30 min at 37'C. PI was added 5 min
prior to the microscopie examination. Intact plasma membrane of whole
cells excluded PI (A). Cells were recognized by an outline trace from the
carboxy-DCF fluorescence (8). PI was obsenred to label DNA of cells
permeabilized by digitonin (C)- Oxidized carboxy-DCF was distributed to
the cytoplasrn of whole cells (8) and retained in organelle-like structures of
perrneabilized cells (D). Images were collected at 63x magnification-
Mitochondnal membrane potential and ROI.
Using the mitochondrial
membrane potential sensitive probe CMX-ros in combination with the ROI probe.
carboxy-DCF. whole celis and permeabilizeû cells had localkation of ROI
staining in the mitochondria (Cl Dl Figure 2-7). Localization of ROI to the
mitochondria in whole cells was not apparent (A. B. Figure 2-7).
WHOLE CELLS
A CMXRos
!
1
i
1
1
1
I
!
I
DIGITONIN PERMEABILIZED CELL
Figure2-7. Fluorssetnce images of OCI/AML-2 with the probes for mitochondrial
membrane patential (CMXRos) and ROI (carboxy-DCF). Whole cells (A and B),
and digitonin petmeabilized cells with the addition of succinate (C and D) were
incubated with the probes for 30 min at 37-C. Oxidized carboxy-DCF (0)was
localized to the energized mitochondria (C) in pemeabilized cells. Images were
collecteci at 6 3 x magnification.
Analpis of Mitochondrial ROI and AY,
2.4.5
in Pemeabiliud Celk by
Flow Cytometry
Cells pemeabilized with digitonin were incubated simuitaneously with the
vanous mitochondrial substrates and inhibitors, and stained with carboxy-DCF
and DilCl(5) for ROI and &Ymlabeling, respedively. Approximately 10,000 cells
were analyzed for each treatment
Figure 2-8 shows representative flow
cytometric histograms for ROI (A) and A T m (8) labeling of perrneabilized cells
with no addition of substrates (shaded region) and in pemeabilized cells
incubated with the succinate, the respiratory substrate for complex II (unshaded
region).
lncubation with succinate decreases the average mitochondnal ROI
and increases the average of AYm.
RESPIRATORY CHAIN ACTlVlTY IMTH
SUCCINATE IN DIGITONIN PERMEABlLlZED
OCI/AML-2
k
2
3
O
O
J
W
O
1
1
ROI
Figure 2-8. Representative histograrns of ROI and mitochondrial membrane potential
( A Y - ) in OCVAML-2 cells. Cells were incubated with the probes carboxy-DCF for ROI,
and DilCl(5) for ALY_ in digitonin pemeabilizedcells in the absence (shaded region), or
presence of sudnate in respiratory buffer for 30 min at 37%.
Approximately 10,000 cells were analyzed
Response of the mitochondrial respiratory chain activity to the various effectors,
as assessed by generation of membrane potential (A',)
and the generation of
ROI. is s h o w in Figure 2-9. Pemeabilized cells mai maVglu and succinate,
which donate electrons to complexes I and II respedively, showed lower ROI
generation and a higher A Y m than cells with no substrate added.
High ROI
generation and low AYm were observed when perrneafized cells had
no
substrate added, or were incubated with antimycin A, an inhibitor for wmplex III,
or with the uncoupler CCCP. The generation of ROI was significantly higher
(ps0.05) with the combined addition of succinate and antimycin A, campared to
succinate alone, consistent with superoxide generation at the complex III site.
MiTOCHONCRlALRESPIRATORY CHAIN
ACTMTY AND ROI GENERATION
O
WOLU
SUC
M
SUC+M
CCCP
EFFECTORS
Figure 2-9. Mitochondrial membrane potential (AY ) and ROI generation were
assessed in cells treated wioi substrates to cornp& I (malate/gfutamate) and to
camplex II (succinate); antimycin A (AA), inhibitor of complex III;and the uncoupling
agent, CCCP. Cells were then incubated with the probes carboxy-DCF for ROI and
DilCl(5) for AY-and analyzed by flow cytometry. Values were calculated from three
separate experirnents and expressed as mean* SEM.
2.5
DISCUSSION
This chapter describes the development of a flow cytometry-based
method to characterire the mitochondrial respiratory chain acüvity in digitonin
permeabilized cells labeled with fluoresenœ probes for mitochondrial membrane
potential (&Ym)and ROI. The method allows for the simuttaneous assessment of
mitochondrial ROI generation and respiratory chain activity in singk cells.
Experirnents were perfomted to measure cellular respiration and ATP
synthesis to confimi the functional integrity of the respiratory chain under the
conditions used. Plasma membrane integrity and labeling with the fluorescence
probes for ROI and AYm were visualized by wnfocal microswpy. This technique
showed that the esterified probe carboxy-DCF diacetate could be loaded into the
mitochondria of digitonin permeabilized cells, where it was retained following
oxidation by ROI.
The data show wnsistencies between the flow cytometry method and the
methods previously used by others to study respiratory chain activities, such as
measuring respiration rate and ATP synthesis (9-1 1). Respiration measurement
in permeabilizedcells shows functional integrity of mitochondria. illustrated by the
oxygraphic trace in Figure 2-3. Followïngpermeabilkation, oxygen consumption
drops to a minimum due to loss of endogenous substrates. The addition of
mallglu, a source for intrarnitochondrial NADH which provides reducing
equivalents to complex 1, or succinate, which provides reducing equivalents to
complex II, induces cells to respire, consuming O2 in the final readon at complex
IV (cytochrome oxidase). The energy released from the passage of electrons
through the respiratory chain at complex 1, III and
the m itochondrial matru< spaœ.
N is used to pump H' out of
This creates an eledrochemical gradient
composed of the proton gradient and the AT,.
The energy stored by this
electrochemical gradient is used to drive the synthesis of ATP. Consistent with
the biochemistry, the synthesis of ATP in the peneabilized cells occurs with the
addition of mallglu, or of succinate in the presence of ADP (Figure 2-5). ADP is a
substrate for ATP synthase, fonning AIP in a condensation reacüon of ADP and
Pi. The addition of ADP alone also generated ATP, indicating the presenœ of
residual respiratory chain substrates in the mitochondrial matrix spaœ. In the
absence of reducing equivalents which occurs upon pemeabilization of celk,
respiration œases, indicating a lack of electron flow through the protein
respiratory chain complex and there is no ATP production. Dissipation of the A Y
m
by the uncoupler CCCP also inhibits synthesis of ATP, but yields a high rate of
oxygen consumption and ROI generation as a consequence of the uncoupling of
the respiratory chain.
Flow analysis of mitochondrial ROI and AY,
generation r e M s the
consequence of electrons shuttling through the respiratory chain. The highest
ROI generation was observeci in the absence of reducing equivalents, and in the
presence succinate with antimycin A, or with the uncoupler, CCCP (Figure 2-9).
The blockage of the respiratory chain by antimycin A prevents the generation of
a ATrn by succinate. Instead, ekctrons accumulating at complex III are accepted
by O2 to forrn superoxide as shown by the increase in DCF fluorescence.
Incubation with reducing equivalents alone yielded lower ROI generation as a
consequence of electrons supporting oxidative phosphorylation, as indicated by
the generation of AYm and ATP synthesis (Figure 2-5).
The images collected by confocal microscopy (Figure 2-7) confimi that
ROI labeling is localized to mitochondrial membrane potential staining. The ROI
generated by other cellular sources was not detected since oxidized DCF is not
retained in the cytoplasm. This flow cytometry-based method therefore facilitates
the shrdy of mitochondrial ROI genemon during apoptosis by: (1) eliminating
background due to other cellular sources of ROI; (2) allowing
biochemical
manipulations of the respiratory chain to localized sources of ROI production. As
with flow cytometry methods generally, the study of cellular heterogeneity may
also be assessed. This is important for the study of apoptosis, because in dnigtreated cells typically only a sub-population is undergoing apoptosis at any
particular time.
2.6
REFERENCES
Foreman HJ, Boveris A Superoxide radical and hydrogen pemxide in mitochondria. In:
Free Radicals in Biology, V (Pryor WA ed.), Academic Press, New York, 1982, pp 65-90.
Wallace DC. Miihondrial Diseases in Man and Mouse. Science, 283: 1482-1487, 1999.
Garcia-Ruiz C, Colell A, Morales A, Kaplowitz N, Femandez-Checa F, Role of oxidative
stress generated fmm the mitochondrial elecbon transport main and mitochondnal
glutathione status in loss of mitochondrial function and
of transcription factor
nuclear factor-uB: Studies with isolated mitochondria and rat hepatocytes. Molecular
PhamacoIogy, 48: 825-834.1995.
Kruidering M, Water B, Heer E, Muîder GJ, Nagelkefke JF. Cisplatininduced nephrotoxicity
in porcine proximaf tubular cells: mitochondrialdysfunction by inhibition of complexes I to IV
of the respiratory c h a h The Joumal of Phannacologyand Expetimntal Therapeutics,
280(2): 638-649, 1997.
Schulze-Osthoff K, Beyaert R, Vandevoorde V, Fiers GH, Fiers W. Depletion of the
mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF.
The EMBO Joumal, 12(8): 3095-3104, 1993.
Hennet Tl Richter Cl Peterhans E- Tumour necrosis factoru induces superoxide anion
generation in mitochondria of L929 cells. BkxhemistryJoumal, 289: 587-592, 1993.
Jia L, Allen PD, Macey MG, Grahn MF, Newbnd AC, Kelsey S. Mitochondriaf electron
transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in
human leukaemic cells: a possible novel mechanism of regulating dmg resistance. BnfiSh
Joumal of Haematology, 98: 686-698, 1997.
Marchetti Pl Susin SA, Decaudin D. Gamen S, Castedo M, Himh Tl Zamzami N, Naval J,
Senik A, Kroemer G. Apoptosis-associated derangement of mitochondrialfunction in cells
lacking mitochondrial DNA. Cancer Research, 56: 2033-2038,1996.
Higuchi M, Honda 1,Proske RJ, Yeh ETH. Regulation of reactive oxygen species-induced
apoptosis and necrosis by caspase 3-like proteases. Onmgene, 17: 2753-2760,1998.
Jia L, Stephen M, Grahn MF, Jiang X-R, Newland AC. lncreased activity and sensitivity of
mitochondrial respiratory enzymes to tumor necrosis factor a-mediated inhibition is
associated with increased cytotoxicity in drug-resistant leukemic cell Iines. Blood, 87(6),
2401-2410, 1996.
Granger DL, Lehninger AL. Sites of inhibition of mitochondrial electron transport in
macrophage-injured neoplastic cells. The Joumal of CeIl Bblogy, 95: 527-535, 1982Hu ZB, Yang GS, Li M, Miyamoto N, Minden MD, McCulloch EA. Mechanism of cytosine
arabinoside toxicity to the blast cells of acute myeloblastic leukemia: involvement of free
radicals. Leukemia, 9: 789:798, 1995.
13. Vercesi AE, Bernardes CF, Hoffmann ME, Gadelha FR, Docampo R. Digitonin
pemeabilùation does not affect mitochondria funcb'on and allows the detemination of the
rnitochondrial membrane potential of Ttypanosma cmi in situ. The Joumal of Biological
Chemistry,266(22): 14431-l44W,l991.
14. Zuurendonk FF, Tager JM- Rapid separation of particulate components and soluble
cytoplasrn of isolateci rat-liver cells. Bbchimr'ca et Biophyska Acta, 333: 393-399, 1974.
15. Rice JE, Lindsay JG. Subcellular fractionaüon of rnitochondria. In Subcellular Fmctionatbn:
a pmctical appmach (Graham JM, R i c h m d 0, eds), Oxford University Press, New York,
1997.
16, Boag JW. Oxygen diffusion and oxygen depletion problems in radiobiology. Cumnt Top*
in Radiation Research Quateriy, 5: 141-1 95, 1969.
17. Dugan LL, Sensi SL, Canroniero LMT, Handran SD, Rothman SM, Lin T-S, Goldberg MP,
Mitochondrial production of r e m e oxygen species in cortical neurons following exposure to
N-methyl-0-aspartate- The Journal of Neuroscience, l5(lO): 6377-6388, 1995.
CHAPTER 3
INVOLVEMENT OF MITOCHONDRIAL ROI GENERATION,
AND THlOREDOXlN IN APOPTOSlS INDUCEO
BY DNA DAMAGING AGENTS
3.1 ABSTRACT
Oxidative stress is associated with the induction of apoptosis by a wide
variety of chernical and physical stimuli. However, the consequences are poorly
understood. The redox balance between the generation of ROI and the adaptiw
response of cells against this oxidative stress has been shown to affect the
sensitivity of cells towards apoptosis. Data are presented showhg
increased
mtiochondrial ROI generation. and the induction of the antioxîdants glutathione
and thioredoxin following treaûnent with the DNA damaging agents ara-C,
etoposide and radiation.
A novel flow cytometry method was used for the
analysis of the mitochondrial membrane potential and ROI generation in digitonin
permeabilized cells 24 hr post-treatment with the DNA damaging agents.
Treated cells still retaining an intact respiratory chain were observed to a have
higher level of mitochondrial ROI generation and a hyperpolarized membrane
potential compared to untreated, control cells when respiration was initiated with
the addition of the malatelglutamate, or sudnate, substrates donating eledrons
to complexes I and II, respectively.
The oxidative stress mediated by the
rnitochondrial ROI was associated with the upregulation of glutathione and
thioredoxin.
Treatrnent with t-BOOH also lead to an increase in thioredoxin
leveis comparable to the DNA damaging agents. These experirnents suggest
that the increase in glutathione and thioredoxin levels might be defensive
responses of cells to the oxidative stress, and consequently may provide a
means for cells to become resistant to drug and radiation treatments.
3.2 INTRODUCflON
The failure of cancer cells to undergo apoptosis is now recognized as an
important mechanism of dnig resistanœ (1-5). Apoptosis is a complex. highly
regulated proœss that remains incampletely understood. However, an extensive
body of literature implicates oxidative stress as a significant mediator of
apoptosis, summarized as follows: 1) exposure to oxidizing conditions, or the
depletion of cellular antioxidants can resuit in apoptosis (6); 2) reactive oxygen
intermediates (ROI) are generated during apoptosis (7, 8, 9); 3) the addition of
antioxidants such as N-aœtylcysteine (26). or the overexpression of antioxidant
enzymes inhibits apoptosis (10). Sinœ oxidative stress can cause upregulation
of antioxidant systems, there is potential for adaptive responses capable of
suppressing oxidative stress mediated apoptosis.
This phenornenon is well
recognized in ischemic heart disease, where heart muscle cells can be protected
frorn reperfimion injury by increased antioxidant enzymes such as MnSOD (11).
However, the importance of this adaptive mechanism in cancer is less well
established.
Apoptosis induced in OCIIAML-2 cells by the DNA damaging agents, araC and irradiation has been previously shown to involve increased generation of
ROI and glutathione (GSH) during the early phase of the proœss. The late
phase of the proœss following the loss of the mitochondrial membrane potential
is characterized by fumer increases in ROI generation and the depletion of
glutathione, indicating a shift in cellular redox balance to an oxidizing state (7, 8).
This rate phase of oxidative stress is most likeiy due to cytochrome c release
from the mitochondrial respiratory chain. resulting in increased mitochondrial ROI
generation (12). These fndings suggest an active mechanism involving ROI
generation and the upregulation of antioxidant systems during ara4 cytoxicity.
This chapter describes experiments to elucidate the mechanisms of oxidative
stress induction in OCI/AML-2 cells during apoptosis, focussing on the role of the
rnbchondrial respiratory chain using the flow cytometry technique described in
chapter 2.
The mitochondrial respiratory chain as a source of ROI generation during
apoptosis has b e n implicated in experiments with ceramide, a putative rnediator
of apoptosis.
lndependent research groups have shown that in isolated
mitochondria, ceramide inhibits the rnitochondrial respiratory chain activity (13)
and stimulates ROI generation (14). However, evidence remains incomplete as
to whether aberrant mitochondrial respiratory chain activity and ROI generation
occur in sifu during apoptosis induction. Data show in this chapter support the
hypothesis that mitochondrial ROI generation mediates the oxidative stress
involved with apoptosis induction by DNA damaging agents. It is also shown that
this oxidative stress is associated with the upregulation of antioxidants,
glutathione and thioredoxin.
The thioredoxinAhioredoxin reductase systern is of particular interest
because it can be upregulated by sub-lethal oxidative stress (15-17). and is able
to suppress apoptosis induced by a wide range of agents including cancer
chemotherapy (18-21).
The role of thioredoxin in apoptosis resuits from its
activity as a redox regulator of protein sulphydryls.
Thioredoxin has high
specificity towards the catalysis of protein disulphide (4-S-) reductions to protein
dithiols (SH)2 (22). Redox regulation of dithiob of the pemieability transition pore
(PT pore) has been demonstrated to be an important factor in the sensitivity of
cells towards apoptosis. Experiments show that oxidation of the PT pore dithiols
causes the pore to be in an open confornation, thus potentiating the apoptosis
process by various stimuli (23-25).
These findings suggest a potential
mechanism for thioredoxin in the maintenance of the PT pore thiol redox state,
thereby inhibiting the pemeability transition.
3.3 MATERIALS AND METHODS
3.3.1 Reagents
Malate, glutamate, succinate, antimycin A,
chlorophenylhydrazone
(CCCP),
sucrose.
carbonyl cyanide m-
KH2PO4. MgC12,
potassium
morpholinopropane sulphonate (MOPS), adenosine S'diphosphate (AD?).
ethylenediaminetetraacetic acid (EDTA), essentially fatty acid ftee bovine semm
albumin (BSA). propidiurn iodide (PI), and digitonin, were purchased from SigmaAldrich
Canada
monobromobimane
Ltd.
(Oakvilk,
(MBrû),
ON).
The
carboxy-DCFDA
ATP
detemination
kit,
(carboxy-dichlorofluorescin
diacetate), chloromethyl-X-rosamine (CMXRos), and DilCI(5) (Ill
',3,3,3',3'hexarnethylindodicarbocyanine) were obtained from Molecular Probes Inc.
(Eugene, OR). Ceramide (C2) was purchased fmm BIOMOL (Plymouth Meeting,
PA).
3.3.2 Cell Culture
The OCVAML-2 cell Iine derived from a patient with acute myeloid
leukemia was obtained from Dr. McCulloch at the Ontario Cancer lnstitute (26).
Cells were grown in a-rninimal essential medium (a-MM), supplemented wÏth
10% fetal bovine serum (FBS), and maintainecl at 37OC. under a humidifid
atrnosphere of 95% air and 5% CO2. Celk were grown at a concentration of
0 . 5 ~o6
1 to 2x1d cellslml, and routinely subcultured every 2-3 days. They were
discarded every three months and reestablished from m e n stock.
3.3.3 Drug and Radiation Treatrnent
Cells were treated during the exponential phase of growth with the
following agents: 7 pM ara-C, 5 p M etoposide, or 7 Gy using a '"CS y-ray un*
Assessrnent of toxicity and oxidative stress were done 24 hr post-treatment.
3.3.4 Flow Cytometry Analysis of Oxidative Stess, AY, and Plasma
Membrane lntegrity
Staining methods. These were as previously described (7, 8). Oxidative
stress was assessed by the simuitaneous measurement of ROI and GSH levels.
The generation of ROI was measured with carboxydichlorofluorescin (carboxyDCF) diacetate.
This probe is retained in celk following hydrolysis of the
diacetate by esterases, and oxidired to a green fluorescent product by ROI.
Levels of GSH were measured with the non-fluorescent sulphydryl probe
monobromobimane (MBr8). This compouds binds specifically to S H groups,
producing bright flouresœnt conjugates. The AYm was measured by staining
with
the
fluorescent
hexamethylindodicarbocyanine).
cyanine
The
dye
plasma
DilCl(5)
(1,l 1,3,313',3'-
membrane integrity was
determined by adding the intercalating DNA stain propidium iodide (PI), which is
excluded by an intact cell membrane. A sample of cells was taken 24 hr posttreatment and stained with 5 pM carboxy-DCF diacetate, and 40 nM DilCl(5) at
37% for 30 min. Five minutes pnor to fiow analysis, 40 pM MBrB and 10 pglml
PI were added to the samples.
Mitochondrial respiratory chah actjvity by ROI and dYm. The flow
cytometry method used to assess respiratory chain function was described in
Chapter 2.
A sampk of cells was resuspended in an equivalent volume of
respiratory buffer consisting of 0.3 M sucrose, 2 mM KH2P04, 5 mM MgC12, 1 mM
EDTA, 0.1% BSA, 1 mM ADP and 20 mM MOPS (pH 7.4). Permeabilization of
the plasma membrane was by addition of 8 pg digitoninfml for 4 min at room
temperature. The respiratory chain acüvity was then assessed selectively by
incubation with the difFerent respiratory chain substrates. 5 mM malate/5 mM
glutamate (mailglu) were used to generate intramitochondrial NADH, a carrier of
electrons to complex l (NADH dehydrogenase).
Electrons to wmplex II
(succinate dehydrogenase) were provided by 5 mM succinate. A sample aliquot
was incubated wiai 0.5 mM carôonyl cyanide m-chkrophenylhydrazone (CCCP).
The probes for ROI and AYmwere added simuftaneously to each sample and
analyzed by flow cytometry after incubation at 37% for 30 min.
Flow cytometry set-up. A Coutter Epics EIite flow cytometer (Coulter,
Florida) was used to analyze the cells stained with the fluorescent probes. This
instrument has three air-cooled lasers emitting Iight at 325, 488 and 633 nm.
The ROI probe, carboxy-DCF, was excited using an argon laser emitting ligM at
488 nm, and the fluoresœnt emission was measured using a 525 nm
+ 10 nm
bandpass filter. Propidium iodide was also excited at 488 nm, and emission was
measured at 640 nm I10 nm. The glutathione probe. MBrB was excited by a
+
325 nm HeCd laser. and a 440 nm 20 nm bandpass filter was used to collect its
fluorescence.
DilCl(5) was excited at 633 nm by a HeNe laser, and
+
fluorescence was measured at 675 nm 20 nm.
3.3.5 Thioredoxin Measuremenb
The intracellular antioxidant protein thioredoxin was measured by flow
cytometry using a
fluoresœinconjugated monoclonal antibody against
thioredoxin. Cells were incubated with the various treatntents for 24 hr. An
aliquot of approximateiy 2x1o6 cells was then taken from each treatrnent, washed
l x in PBS, then fixed and penneabilized using the CytofixlCytoperm Kit
(PharMingen Cat. # 2075KK). The protocol wnsisted of fixing the cells in a
solution of 4% parafomaldehyde at 4OC for 20 min. Cells were then washed in a
staining buffer, and resuspended in 50 pl penneabilization bmer with a
fluorescein-conjugated anti-human thioredoxin monodonal antibody at a
concentration of 9 pglml. This antibody, a clone binding to full length thioredoxin
(27), was provided by Dr. Charies Shih (Phamingen, San Diego, California).
The fluorescein was excited at 488 nm, and fluorescence was measured using a
525 nm I10 nm bandpass fiiter.
3.4 RESULTS
3.4.1 Drug Toxicity Characdsrizeâ by L o u of Plasma Membrane Integrity
and A Y / ,
The effects on plasma membrane integrity and mitochondria! membrane
potential (AYm) of cells after 24 hr treatment of cells with 7 pM ara-C. 5 PM
etoposide and 7 Gy y-radiation were analyzed by flow cytometry, and results
shown in Figure 3-1. As shown in the top row the treatrnents resuited in sirnilar
percentages of cells retaining an intact plasma membrane, with approximately
30% of cells treated with the DNA damaging agents showing a disrupted plasma
membrane in cornparison to control. Bivariate plots in the bottom row show that
cells retaining an intact plasma membrane consist of a population with a high
A Y (A),
~ similar to untreated control cells, and a population of low A T m (B). This
indicates that the sequence of events involves the loss of the AT,,
prior to the
loss of surface membrane integrity. This is consistent with the apoptotic model
which proposes that damage to the mitochondrial respiratory chain following the
release of cytochrome c into the cytoplasm iniüates the final events of cell
destruction. lrreversible cell damage is observeci in population C, characterized
by a low AY,, and a niptured plasma membrane, indicated by cell pemeability
towards the dye PI.
CONTROL
7 pM ARA-C
l2 ,
-
O
.
3
0
.
5 fl ETOPOSIDE
7 GY
PLASMA MEMBRANE INTEGRITY
Figure 3-1.Cytutoxic effects of DNA damaging agents- Assessrnent of celt plasma
membrane integrity (PI), and AT- Mer 24 hr treatment with ara-C, etoposide and
radiation. The cursors on ttie top row histograms show percentage of cells with
an intact plasma membrane. The bottom row show cytotoxic effects of treatments
result in similar patterns of cells having two defined populations (B.C).
3.4.2
Involvement of Oxidative Stmm in Early and Late Phases of
Apoptosis
The results presented in Figure 3-2 show that after 24 hr treatment with
the DNA damaging agents, cells undergo similar oxidative stress changes. Cells
excluding PI are characterized in bivariate plots that demonstrate both increased
ROI generation and glutathione early in the apoptosis stage (population A). Cells
in the later stages of apoptosis, characterizad by the loss of A%.
show further
increases ROI generation and a depletion of glutathione (population B). Cells
undergoing the intemiediate stages of plasma membrane dis~ptionare leaky
toward the intracellular probes as characterized by population C, and celk with
decreasing levels of glutathione in population B (bottom row). These results
show that oxidative stress during apoptosis is a m p l e x phenomenon that is
apparently linked to aiterations in mitochondria.
CONTROL
7
ARA-C
5 PM ETOPOSIDE
REACTIVE OXYGEN INTERMEDIATES
Figure 3-2. Oxidative stress in early and late stages of apoptosis. Bivariate plots of
flow cytometry analysis of reactive oxygen intermediates, and glutathione against
mitochondrial membrane potential (AT-) were made in cells af€er24 hr treatrnent with
ara-C, etoposide and radiation.
The oxidative stress pattern is similar between the agents even though they have
different modes of action. An analysis of ROI and glutathione (GSH) levels in
cells maintaining a AYm after treatrnent to the various DNA damaging agent is
shown in Figure 3-3.Values are mean ISEM of three separate experiments.
Treatment with the D M damaging agents resuited in an increase in
generation of ROI and glutathione early in the apoptosis defined by the
population of œIJs that exclude PI and maintain an energized mitochondrial
membrane potential (population A. Figure 3-2). Irradiation and etoposide
significantly stimulated ROI generation compared to control. untreated cells
(peO.05). The increase in GSH was greatest wial etoposide treatrnent and is
significantly higher than control (pc0.05).
The involvement of ROI generation and the induction of GSH during
apoptosis followïngexposure to a variety of DNA damaging agents suggests that
these agents are triggering e common apoptosis pathway that involves oxidative
stress. These results are consistent with previous work done by ouf labotatory
with the agents ara-C and radiation (7. 8).
INDUCTION OF ONDATIVE STRESS
BY ûNA DAMAGING AGENTS
Figure 3-3. Effecb of treatments on levels of ROI and GSH after 24 hrs
in cells early in the apoptosis induction phase, prior to the loss of the
mitochondrial membrane potential. The asterisk indicate statistical
significance (pcJ.05) compared to control, untreated cells.
3.4.3
Assessrnent of Mitochondrial ROI and A T m During Drug and
Radiation Treatment
The mechanisrn of ROI generation early in the apoptosis stage. pnor to
the loss of the rnitochondrial membrane potential @Y,,,) is of particular interest for
the following reasons: first, in the role of mediating apoptosis; secondly, in the
role of mediating cell suMval, where ROI generated during apoptosis are
associated with the activation of defense mechanisms against the oxidative
stress (7, 8, 28). The flow cytometry method described in chapter 2, was used to
elucidate whether mitochondrial respiratory chah activity and ROI generation are
compromised during apoptosis induction by the DNA damaging agents.
Mitochondrial respiratory chain activity was assessed in samples of cells
24 hr post-treatment, based on preliminary time course experiments.
Four
conditions were examined, the effects of digitonin pemeabilization of plasma
membrane alone with no addition of substrates, or
with the addition of the
respiratory chain substrates malate/glutamate, or succinate, or with the
uncoupler CCCP. Figure 3 4 is a representative bivariate plat of mitochondrial
membrane potential (ATm) and mitochondrial ROI generation under the various
mitochondrial effectors in control, untreated cells and cells treated for 24 hr with
ara-C.
NO SUBSTRATES
MAUGLU
SUC
UNCOUPLER
CONTROL
REACTIVE OXYGEN INTERMEDIATES
Figure 3-4. Mitochondnal respiratory chain adivity in cdls 24 hr pst-treatrnent with
ara-C. Representative bivariate plots of generation of ROI and A I in digitonin
permeabilized cells with the various respiratory chain effectors: cornplex I substrates.
maiatelglutarnate. the the cornplex II su-bstrate,suaïnate, and the urkupling agent.
CCCP.
Digitonin perrneabilüation of cells with no addition of substrates, or with the
addition of the uncoupling agent. CCCP resuited in a homogenous population of
cells after treatment with araC, etoposide or radiation. similar to control.
untreated cells.
However, when dnig treated cells were incubated with the
respiratory chain substrates, a separation of sub-populations, characterized by
differences in membrane potential, was observed (B and E. C and FI bottom
row). The proportion of cells that responded to generate a AYm (E and F) was
similar to the proportion previously found to have energized mitochondria in the
intact cell assay, shown as population A in Figure 3-2. Similar bivariate plots
were observed for etoposide and radiation treatments. For the assessment of
the ROI generation early in the apoptosis stage, only cells which could generate
a AY, with the addition of the respiratory substrates were analyzed.
Mr'OCHONDRlAL ROI GENERATION IN AML-2
POST TREATMENT 24 HR
NOSUBSTRATES
kUUGLU
SUC
MITOCHONORIAL RESPIRATORY CHAlN EFFECTORS
Figure 3-5. lncreased mitochondrial ROI generabion in cells undergoing
the early phase of apoptosis after 24 hr treatment with various DNA
damaging agents. Assessrnent was made under the selective conditions
of substrate addition to complexes 1, or II,or in the presence of the
uncoupler CCCP in digitonin permeabilizedcells. Values are mean fSEM.
The mean I SEM values of three separate experiments of ROI are shown in
Figure 3-5, and of A Y m are shown in Figure 3-6. There was a statistically
significant increase mitochondrial ROI generation in al1 conditions assessed,
(pc0.05) compared to untreated control cells. Aberrant respiratory chain activity
was also observed. as assessed by the generation of
Figure 3-6.
the
AYm as shown in
NO SUBÇTRATES M U U
SUC
UNCOUPLER
MITOCHONDRIAL RESPIRATORY CHAIN EFFECTORS
Figure 3-6. Hyperpolarizationof mitochondrial membrane potentiai in cells
undergoing the early phase of apoptosis after 24 hr treatment with various
DNA damaging agents. Assessrnent was made under the selective conditions
of substrate addition to complexes 1, or II. or in the presence of the uncoupler
CCCP of digitonin permeabilized cdls. Values are meanf SEM.
The addition of substrates to cornplex I, maUglu. or succinate, a substrate for
complex II produced a statistically signifiant hyperpolarked A Y ~
in drug and
radiation treated cells compareci to untreated controls.
The results show in Figure 3-5 and Figure 3-6 demonstrate the
involvement of the respiratory chain in the generation of ROI, and
hyperpolarization of the inner membrane early in the induction of apoptosis
following exposure to the DNA damaging agents, as was previously obsenred in
the analysis of Mole cells by flow cytometry (top row. Figure 3-2). These
observations are consistent wÏth the idea that mitochondrial aiterations play a
role in the eariy stages of cell death. in addition to
the later events that are
and
, the release of cytochrome c (12).
associated with the loss of AT,,
Ceramide. Experiments have demonstrated that ceramide, a putative
mediator of apoptosis, c m inhibit the advity of the respiratory chah cornplex III,
and can induœ ROI generation in isolated mitochondria (13, 14). Since ara-C
and radiation have both k e n shown to increase ceramide proâuction (36, 37),
effects of ceramide on mitochondrial ROI and AYm were assessed using the
digitonin permeabilizationtechnique, as shown in Figure 3-7.
EFFCTS OF CERAMIDE ON MiTOCHONDRlAL
RESPIRAlûRY CHAIN ACmVïiY
SUC
SUC+M
SUC+CER
UNCOUPLER
MITOCHONORIALRESPIRATORY C W N EFFECTORS
Figure 3-7. Eff6cts of ceramide on the mitochondrial generation of ROI
and A T - in the presence of succinate, the substrate to respiratory chain
complex IIcornpared to the addition of succinate alone, or with the
complex III inhibitor antimycin A (AA). Statistical significance as cornpared
to induction of respiratory chah activ'ï by succinate is indicated by the
asterisk (pe0.05). Values are mean* SEM of three separate experirnents..
The effeds of ceramide on the respiratory chain acüvity during respiration in the
presence of succinate were wmpared to the wmplex III inhibitor, antimycin A
(AA). Treatrnent with 10 UM ceramide blocked the respiratory chain activity in a
similar pattern to AA, showing a decreased A Y m and an increased
generation.
ROI
These results are similar to those previously reported (13, 14).
although they are not entirely consistent with the alterations obsetved early in the
apoptotic phase in œfls treated with the DNA damaging agents, where the
effects of these agents result in a hyperpolarization. rather than a loss of AYm.
3.4.4
Thioredoxin Expression During Drug and Radiation-înduced
Apoptosis
As shown in Figure 3-3, oxidative stress following treatment with ara-C,
etoposide and radiation is initially associatecl with the induction of the free radical
scavenger glutathione. Accumulating evidenœ suggesb that systems involved
with the maintenance of the redox states of mitochondrial sufphydryls play an
important role in determining the sensitivity of cells toward apoptosis (23-25).
Thioredoxin (TRX) has been previously shown to be inducible under a variety of
oxidative stress stimuli including hydrogen peroxide, irradiation, hypoxia, and
ischemia (23-25). It was therefore of interest to detemine if the cellular content
of TRX was affected by oxidative stress following drug and radiation treatments.
mmtion of the Fluomsc8in-mAb anti-thbmdoxin. In order to optimize the
immunofiuorescenœ staining method for TRX, an antibody dilution cunre was
prepared using fixed, penneabilized OCVAML-2 cells, shown in Figure 36.
TlTRATlON CURVE FOR FITGmAb
AGAlNST THIOREDOXIN
100
FffC-mAb AGAlNST THlOREOOXlN (pghnlj
Figure 3 8 . Cells were fixed, permeabilized and stained at increasing
antibody concentrations. and analyzed by flow cytorneûy.
A linear relationship was observed for the staining of the fluoresœin-mAb antithioredoxin in the range of O to 20 pglml of the antibody. Near saturation for the
antibody staining was reached beyond 20 pglml.
An antibody staining
concentration of 9 pglmf was used for assessrnent of TRX expression. This
concentration was just below the saturation to minimize effects of non-specific
binding.
Measunng dhiomdbxin during apoptosis inducf!onn Thioredoxin expression
after 24 hr treatment with the DNA damaging agents was analyzed by fiow
cytometry. A representaüve histogram of the effeds on thioredoxin levels posttreatment with etoposide is shown in Figure 3-9. The left panel shows a bimodal
distribution of thioredoxin with etoposide treatment. wmpared to untreated,
control cells (shaded region). The conesponding light scatter changes, indicative
of cefl morphology. on
the panels to the right show that cells expressing higher
thioredoxin levels than control have light scatter patterns similar to control cells.
Figure 3-9.Alteration of AML-2 cellular thioredoxin with 5 pM etoposide
treatment compared to untreated control cells (shaded region, left panel).
Cells expressing high thioredoxin (top, right panel) have light scatter patterns
that are similar to mntrol cells. Lower thioredoxin expressing cells with
etoposide treatment (bottom, right panel) have light scatter patterns
which are characteristic of cytoplasmic shrinkage and disniption of plasma
membrane.
Cells expressing lower thioredoxin than controls have light scatter patterns that
are associated with œlls that are more damaged. This population was excluded
from the subsequent analysis of thioredoxin expression 24 hr post-treatment, as
the interest is in the regulation of such antioxidant systems in the eariy phase of
apoptosis, pnor to ineversible commitment to cell death. Treatments with the
DNA damaging agents resulted in increased levels of thioredoxin protein as
s h o w in Figure 3-10.
In the case of etoposide. the increase was *sücalv
significant compareci to untreated. m t r o l cells (pe0.05).
Treatment with the
oxidant tert-BOOH resulted in an increase comparable to the cytotoxic agents.
This is consistent with experiments showhg induction of thioredoxin mRNA in
cells after treatment with oxidative stress associated stimuli (e.g. radiation,
hypoxia and ischemia) (15, 16, 28).
INCREASED THIOREOOXIN EXPRESSION
wrrH OXIDATNE ~ E S ASSOCIATED
S
TREATMENTS IN OCUAML-2
50
meant SEM
*
T
3.5
DISCUSSION
The data presented in this chapter show that apoptosis induction with the
DNA damaging agents, ara-C, etoposide and y-radiation is associateci oxidative
stress similar to previous published work, chancterüed by increased ROI
generation and glutathione in cells early in the stages of apoptosis (7, 8).
Experiments investigating the mitochondrial respiratory chain as the ROI
generating source were then perfoned using the fbw cytometry meaiod
described in chapter 2 for the analysis of the respiratoiy chain activity in digitonin
perrneabifized cells. The data show that in the early phases of apoptosis, cells
treated with the DNA damaging agents retain an intact respiratory chain. and
have higher mitochondrial ROI generation compared to control. untreated cells.
This increase in ROI was associated with the sub-population of treated cells
maintaining a hyperpolarized membrane potential seen under respiration induced
with addition of respiratory chain substrates both to complexes I and II, as
assessed in pemeabilized cells. Data were also presented to support previous
findings that this oxidative stress is associated with the induction of cellular
antioxidant defense systems. Treatrnent with the DNA damaging agents resuîted
in the upregulation of glutathione and thioredoxin. lncreasing evidence suggests
that the redox balance between prooxidants and antioxidants are important
deteminants of the sensiüvity of cells towards apoptosis.
These findings
tharefore suggest that cells might be capable of adapting to the oxidative stress
by upregulating defensive systems that favour cell survival.
Previousîy reported experiments investigating mitochondrial respiratory
chain aiterations dunng apoptosis have shown that aiterations in apoptotic
sensitivity can occur with addition of respiratory chain inhibitors, or by the
elimination of a fundional respiratory chain (38). However, results from other
groups using similar methods have disputed the involvement of mitochondrial
ROI (39, 40).
These earlier studies failed to assess mitochondrial ROI
generation during apoptosis n
i ducto
in
in situ. The advantages of using the flow
cytometry method described in chapter 2 are that it excludes other cellular
sources of ROI, and allows the direct assessrnent of respiratory chain activity
including ROI generation during the course of apoptosis.
Furthemore, the
technique is able to distinguish between cells in the eariy phases of apoptosis
from those that have lost mitochondrial membrane potential.
Mitochondnal ROI generation prier to the loss of mitonchondrial
membrane potential. Using the flow cytometry method to assess respiratory
chah activity in digitonin pemeabilized cells, cytotoxic treatments by ara-C,
etoposide, and radiation similady increased mitochondrial ROI and produced a
hyperpolarked A-
with the addition of maUglu, substrates for complex 1, or
succinate, the substrate for wmplex II of the respiratory chain. The membrane
potential is generated when electrons are passed through complexes 1, IIIand IV
of the respiratory chain, and is dissipated by ATP synthase in the production of
ATP. Thus, the hyperpolarized AYm observed following dnig treatment suggests
elher an impairnent of the electron transport downstream of both complexes I
and II, (since maVglu and succinate are both able to support a AYm), or the
inhibition of ATP synthase which, under normal activity, uses the electrochemical
gradient for ATP production.
In either situation, the consequenœ is an
accumulation of electrons along the respiratory chain, thus promoting the
generation of ROI at sites in complexes that can donate single electrons to
molecular oxygen.
The putative apoptosis mediator ceramide has been previously associated
with inhibition of the respiratory chain at compiex III, and able to stimulate
generation of ROI (13, 14). This is consistent with the abifity of respiratory
substrates to both complex 1, and complex II to generate ROI, as the eiectrons
acquired by either system are blocked by impairement of a downstream site (e-g.
complex III).
However, if ceramide is indeed an apoptosis mediator for the
agents used to trigger apoptosis in this study, then the ROI generation would be
expected to occur early in the induction phase. Although ceramide had no
significant effect on mitochondrial ROI generation in control cells, it effectively
inhibited the generation of a AY,,,, suggesting that ceramide might induœ the PT
pore opening by depolarization of the mitochondrial transmembrane potential.
Depolarization of AY, has been s h o w to be one of the triggers of PT pore
opening (41). Ecperiments showing the release of cyt c without the concomitant
loss AYm also suggest that the ROI generation by the mitochondrial respiratory
chain can occur in cells that are slowly losing cyt c by mechanisms such as
transient opening of the penneability transition pore, or channels involving proapoptotic mernbers of the Bcl-2 family, such as Bax (29-31).
Recently, an alternative mechanism for mitochondnal ROI generation has
been proposed that involves the adenine nucleotide translocator (ANT)
component of the permeability transition pore (32). The madel shows that during
apoptosis induction, the failure of the ANT prevents the exchange of ADP for
ATP into the mitochondrial matrix space. As a consequence, low ADP levels in
~ the
the matrix space inhibit ATP synthase, resutting in a hyperpolarized A V as
proton gradient created by the mitochondrial respiratory chah is no longer
utilized by ATP synthase for the production of ATP. Hyperpolarization of the
mitochondrial membrane favours the generation of ROI, as the complexes of the
respiratory chain remain in the reduœd state. Overexpression of BcI-x', an antiapoptotic member of the Bcl-2 farnily, sufficiently maintains the exchange of
ADP/ATP during growth factor withdrawal, resufting in the protection of cells.
This modal provides an explanation for the mitochondnal ROI generation and the
hyperpolarizationof the AYmobsewed in cells 24 hr post-treatrnent.
The role of mitochondrial ROI involvement in the induction of apoptosis
has been postulated to be rnediated by oxidation of dithiol groups on the adenine
nucleotide translocatcr
which
causes
the
permeability transition
and
consequently the release of cytochrome c. However, oxidative stress occuring
during apoptosis has also k e n assaciated with the induction of antioxidant
systems which can protect cells from toxic insults (10, 28). This mechanism of
protection has been previously generalized to the wntrol of the intracellular
redox level. Involvement of the PT pore in the apoptotic cascade provides a
particular instance where the antioxidant thioredoxin might prevent the induction
of apoptosis. It has been previously shown that the PT pore opening is more
sensitive to agents that oxidize mitochondrial sulphydryl groups than to fkee
radical generating oxidants (25). Also, agents that maintain the dithiols in a
reduced state inhibit PT pore opening (33). These observations suggest that the
redox regulation of the PT dithiols is an important mechanism detemining cell
survival. The regeneration of protein sulphydryl groups in vivo depends primarily
on the glutaredoxin and thioredonnRhioredoxin reductase systems. Of particular
interest is thioredoxin, as it has higher specificity than glutaredoxin for reduction
of protein disulphides (22).
Thiomdoxin expression during apoptosis induction. Apoptosis induction
resulted in a bimodal distribution of thioredoxin protein expression 24 hr posttreatrnent with ara-C, etoposide and radiation. These thioredoxin changes reflect
the changes in GSH levels during apoptosis induction. The increase in both
thioredoxin and GSH at an eariy stage in apoptosis suggests a wmmon
regulatory mechanism. Sinœ the oxidant t-BOOH also induced thioredoxin, an
oxidative stress mechanism similar to that shown in the ischemic mdels is
suggested (16, 34). Expression levels of mRNA thioredoxin ranging from 1-5 to
10-fold higher than control cells experiments correlated with protection of cells
towards various oxidative stress associated injuries (15, 16, 35). The induction
of thioredoxin protein has not k e n previously reported. However. the previous
data on the regulation of thioredoxin at the level of mRNA are consistent with the
increase in thioredoxin protein seen with drug and radiation induced apoptosis in
OCIIAML-2 cells.
Effects on ROI, GSH, and thiom&xin during apoptosis. Treatrnent of
OCVAML-2 cells with the DNA damaging agents, ara-C, etoposide, and radiation
results in an oxidative stress response involving mitochondrial ROI that occurs
relatively eariy in the apoptotic proces. This ROI generation has now been
shown to resuit from disruption of the mitochondrial respiratory chain activity.
The effect of this on cells may be two-fold. First, mitochondrial ROI generation
might induce an oxidative environment that favours further damage to celb.
Secondly, ROI can lead to the upregulation of glutathione and thioredoxin.
suggesting that the ROI generation acüvates cellular defense mechanisms via an
adaptive response mechanism. Thus, mitochondnal generation of ROI, following
DNA damage wuld potentially promote cell survival, as well as mediating cell
damage.
3.6
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CHAPTER 4
SUMMARY AND FUTURE DIRECTIONS
4.1 SUMMARY
The experiments presentd in this thesis were designed to investigate
cellular redox regulation during apoptosis induced by various DNA damaging
agents (ie. ara-C, etoposide, y-radiation), with particular emphasis on
mitochondnal ROI generation.
Previous work has shown that induction of
apoptosis by ara-C and radiation in OCIIAML-2 cells is associated with the
following changes in cellubr redox levels:
1)
cells retativeiy eady in the
apoptosis induction generated ROI and upregulated GSH levels; 2) cells which
had lost AT,,,, also lost the ability to regulate their redox environment, and
showed a further increase in ROI generation with depietion of GSH; 3) the final
destruction of these cells was characterized by the loss of plasma membrane
integrity and the appearanœ of typical morphologie apoptotic bodies (1, 2). The
loss of mitachondrial membrane potential is considered to indicate irreversible
commitment to cell death, either by apoptosis, or due to biochemical
consequences of the permeability transition, such as failure of ATP synthesis.
Of particular interest is the redox regulation dunng the early apoptotic phase,
prior to the commitment to cell death. The data shown in chapter 3 suggest that
the generation of ROI during this eariy phase has two affects. First, it may be
involved with the induction of the loss of the AYm and consequently a
commitment to the final destruction of the cell. Secandly, it may be involved with
the potentiation of cell survival by the induction of antioxidant defense systems.
The paradoxical roles of oxidative stress have been reported by other groups
showing that the addition of oxidants (e.g.
H202)
can trigger the permeability
transition (loss of ATm) and induœ the morphological features typical of
apoptosis. or that oxidative stress can induœ antioxidant systems (e-g. MnSOD,
thioredoxin) and potentiate cell survival (35).
These findings suggest that the
signals rnediating cell death and cell survival are integrated at the cellular redox
level.
The novel fndings presented in this thesis are firstly. that the oxidative
stress occuring in apoptosis trïggered by the various DNA damaging agents is
due to ROI generation by the mitochondrial respiratory chain. Second, that this
is associated with the upregulation of the redox sensitive protein thioredoxin.
In chapter 2. the development of a novel flow cytometry-based method
was described for the assessrnent of the mitochondrial respiratory chain activity
by the simuitaneous characterization of mitochondrial generation of ROI and
membrane potential in digitonin penneabilized cells.
The characterization of
respiratory chain activity by this flow cytometry-based method was shown to be
consistent with other methods, such as the measuring of oxygen consumption. or
of ATP production. The advantages of this rnethoâ are: 1) Ï t allows the direct
analysis of ROI generation with respect to the various respiratory chain
complexes, as characterized by the generation of mitochondrial membrane
potential under selective respiratory conditions; 2) the respiratory chain activity
can be assessed in individual cells; 3) the method is insensitive to other cellular
sources of ROI generation.
Data presented in chapter 3 show that the eariy induction phase of
apoptosis is associated with similar redox alterations for agents with difVerent
DNA darnaging mechanisms. namely ara-C. etoposide and radiation. Using the
method described in chapter 2. the oxidative stress observed in cells undergoing
the early phase of apoptosis induction was shown to be mediated by increased
mitochondrial ROI generation in cells that still retained an intact respiratory chain
capable of generating a mitochondrial membrane potential. Substrates for both
respiratory chain m mpiex I (malatelglutamate), and for complex II (succinate)
could support the generation of ROI and a hyperpobrized membrane potential.
The mechanism of mitochondrial ROI generation has been previously postulated
to be mediated by inhibition of the respiratory chain complexes. Support for this
cornes fmm experiments done with ceramide, a putative mediator of apoptosis.
Independent groups have shown that effects of ceramide include inhibition of
complex III of the respiratory chain (6), and induction of mitochondrial ROI
generation (7). The effects of ceramide on our model show similar effects on the
respiratory chain activity to those obtained with antimycin A, an inhibitor of
complex III. Addition of ceramide increased rnitochondrial ROI and decreased
mitochondrial membrane potential. Although consistent with data reported in the
literature, these findings do not entirely explain the observation in drug or
radiation treated cells, where high mitochondrial ROI is associated with
hyperpolarization, rather than the loss of mitochondrial membrane potential.
Thus, effects on the respiratory chain during apoptosis induction may be more
complex than has been previously proposed.
Elucidating the mechanisms of mitochondrial ROI genemüon during
apoptosis is of wntinued interest in mure work, particularfy as data in chapter 3
show associated induction of the antioxidants glutathione and thioredoxin. The
novel finding that thioredoxin is increased dunng apoptosis induced by
chemotherapeutic agents (ara*,
etoposide) is of particular interest for the
followhg reasons: 1) increased expression of thioredoxin in transfected œlls
confers resistanœ to chemotherapy agents associated with DNA damage; 2)
thioredoxin can regulate the expression of other antioxidan (e.g . GSH, MnSOD)
(8, 9); 3) thioredoxin can affect activity of proteins central to apoptosis induction.
such as the permeability transition pore (IO); 4) thioredoxin can affect activity of
stress inducible transcription factors associated with potentiating cell survival
(e-g. NF-KB, AP-1) (11). These observations impficate thioredoxin upregulation
to be an adaptive response to the oxidative stress that occurs following DNA
damage. and thus may provide a mechanism for drug-induced resistanœ.
4.2 FUTURE WORK
The generation of mitochondfial ROI, in addition to its involvement
cancer models, has been associated with diseases associated with mitochondrial
DNA mutations, such as aging, myopathies and Parkinson's disease. and in
cellular injury, such as ischemia and hypoxia (12-14).
The flow cytometry
method developed in chapter 2, allows assessrnent of mitochondrial ROI
involvement in such diseases in conjunction with determining abnormal
functioning of the respiratory chain cornplex activity by measuring mitochondrial
membrane potential under selective respiratory conditions.
Projects involving the use of the method have already been initiateci with
Dr. Brian Robinson, elucidating the invohrement of rnitochondrial ROI in inherited
mitochondrial disorders of children characterized by abnormal respiratory chain
function.
Experiments in collaboration
performed to elucidate
with Dr. lona Weir are also being
the rok of rnitochondrial ROI in apoptosis induction in
plant cells treated with camptothecin. Thus, the m e # d is applicable to a wide
range of cell systems for assessing mitochondrial ROI invohrement and aberrant
mitochondrial respiratory chain adivities.
Mechanisms regulating ROI generation are of particular interest as they
can induce cellular defense systems against the oxidative stress and potentiate
cell survival. The induction of thioredoxin during apoptosis induœd by the
chemotherapy agents ara4 and etoposide suggests its involvement in a
mechanism for drug induced resistanœ.
Future projects to examine the
significance of the ROI generated during apoptosis in the regulation of
antioxidants, such as thioredoxin would involve addressing the issues on
different levels.
First, the examination of thioredoxin expression and its
inducibility in other cell lines under various oxidative stresses.
The highest
reported thioredoxin mRNA induction of 14-fold was made in HT-29 colon canœr
cells under hypoxic conditions (4). Secondiy, the examinaüon of the other
members of the thioredoxin system, such as thioredoxin teductase which uses
NADPH to catalyze the reductïon of the redox sensitive sulphydryls on
thioredoxin, and thioredoxin peroxidases which reduœ hydrogen peroxide.
Increased adivity of these enzymes has recently been show to protect cells
from apoptosis assocîated with oxidative stress (15-1 8). Of particular interest in
future experirnents is the inducibility of these proteins and the development of
drug and radiation resistanœ in cancer.
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