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Initiation of apoptosis by application of high-intensity electric fields

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Initiation of apoptosis by application of high-intensity electric fields

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Content Initiation of Apoptosis by Application of High Intensity Electric Fields
by
Clayton Young
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Biomedical Engineering)
August 2002
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U M I N um ber: 1 4 1 4 8 9 3
UMI
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U N IV E R S ITY O F S O U T H E R N C A LIF O R N IA
T H E G R A D U A TE S C H O O L
U N IV E R S IT Y P A R K
LOS A N G E LE S . C A L IF O R N IA 9 0 0 0 7
This thesis, written by
...................................C la y t on Young_________________
under the direction of h£s. Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillm ent of the
requirements for the degree of
..............................................Ma&t.ej?..j?S..££±mce-Bjlp.mediQQl.E.ug.,
........
D ta n
D ate_ _
THESIS COMMITTEE
Chairman
.
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TABLE OF CONTENTS
List of Tables
List of Figures
Abstract
Introduction
Theory-Electrical
Capacitive Membrane Charging
Lumped Circuit Model
Theory-Biological
Apoptosis
Characterization of Apoptosis - Annexin V Assay
Characterization of Apoptosis - Forward and Side Scatter
Method
Cell Line
Pulse Generator
Environment
Cell Suspension Treatment
Flow Cytometric Analysis
Results
Trypan Blue Exclusion
Annexin V-FITC / Propidium Iodide Staining
Discussion
Conclusion
References
References (Alphabetical)
iii
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5
5
7
11
12
15
18
19
20
20
21
23
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26
29
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iii
LIST OF TABLES
Table 1: Approximate dimensions of Jurkat-T lymphocytes and mitochondrion 6
Table 2: Derived parameters for capacitive membrane charging 7
Table 3: Lumped circuit parameters for linear circuit model of a single cell 8
Table 4: Pulse parameters for arbitrary field stimulus to a cell 9
Table 5: Possible outcomes of the Annexin V / PI assay 14
Table 6: Experiment protocol in tabular format 21
Table 7: Experiment result: cell viability count resulting from TBE assay 23
Table 8: Experiment result: percentage apoptotic yield 10/05/01 24
Table 9: Experiment result: percentage apoptotic yield 10/17/01 25
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LIST OF FIGURES
iv
Figure 1: Linear circuit model of a single cell in suspension 8
Figure 2: Simulation result of capacitive membrane charging in a linear circuit model 9
Figure 3: Power spectral density plot of the field stimulus and membrane response 10
Figure 4: Micrograph of an apoptotic cell, showing typical fragmented characteristics 11
Figure 5: Mitochondrial membrane rupturing with release of cytotoxic molecules 12
Figure 6: Dot plot migration of cell populations during execution of the apoptotic 17
cascade analyzed by flow cytometry.
Figure 7: Pulse generator hardware used for pulsing of suspended cells 19
Figure 8: Overview of experiment procedure 22
Figure 9: Apoptotic yields of 10/05/01 experiment in graphical format 24
Figure 10: Apoptotic yields of 10/17/01 experiment in graphical format 25
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V
ABSTRACT
Exposure of suspended cells to high-intensity, pulsed electric fields of
nanosecond-scale duration force a significant percentage of the treated cell population
to undergo apoptosis, or genetically mediated cell death. Field-treated samples show an
increase in the population fraction demonstrating morphological changes to the cell
structure which are indicative of the apoptotic cascade. Flow cytometric inspection also
revealed negligible plasma membrane permeability, implying that field-induced changes
were localized within the intracellular space. Changes to the cell ultrastructure initiated
solely by the electric field stimulus demonstrate the ability to manipulate intracellular
processes noninvasively without the need of exogenous molecules or organisms. The
apoptotic response to externally applied electric fields demonstrate a possible method of
deleting undesired cell populations noninvasively without evoking undesired side-effects
compared to more traditional chemotherapeutic, radiotheraputic, or surgical techniques.
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1
INTRODUCTION
Aqueous pore formation in cell membranes resulting from exposure to externally
applied electric fields has been studied for decades since application of the technique
enables limited control of the barrier function of the outer membrane. The utility of field-
mediated changes to the membrane ultrastructure cannot be understated since the
ability to control the lipid partition implies an ability to manipulate critical cellular functions
that are dependent on the membrane’s ability to separate macromolecules or ions.
Early studies of field-induced changes [1] reported observations of large, yet reversible,
decreases in transmembrane resistance of synthesized membrane structures of planar
geometry. In the study, reversible specific resistances collapsing from .1 GQ*cm2 to 0.4
Q*cm2 was confirmed as transmembrane potentials exceeded threshold voltages of
approximately 1 Volt. Other studies investigating reversible conductances with
application to viable cells [2] reported similar results if cells are subjected to an electric
field with sufficiently high strength and duration. The lethal effects of field-induced
stimulation were also observed as cells rupture and lyse upon prolonged field exposure.
It was [3] theorized that long pulse durations result in an overabundance of aqueous
pores sufficient to irreversibly compromise membrane integrity, resulting in dissolution of
cytosolic fluids while also permitting large osmotic pressure imbalances capable of
rupturing the cell.
It was also theorized [4,5] that the rise in membrane conductivity was largely due to
the presence of aqueous-pores bridging the intracellular and extracellular space,
allowing ions or macromolecules to freely migrate beyond the membrane partition.
Freeze-fractured micrographs [6] of cells, captured shortly after shocking, were obtained
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2
in an attempt to directly observe the membrane perturbations. Relatively large-scale
structural defects clearly enable the diffusion of particles through the membrane.
Asymmetric dissolution of intracellular solutes through field-treated oat protoplasts
further illustrates the concept of field-induced pore formation.
Applications exploiting the reversible membrane permeability phenomenon have
been developed [7, 8]. Introduction of plasmid vectors into the cytosol by membrane
permeabilization has been commercialized to provide a reliable method of
transformation (GenePulser, Bio-Rad). Introduction of exogenous macromolecules prior
to the field-induced pore approach was performed by more laborious techniques, such
as transformation by bacteriophage. More recent developments have focused on the
forced uptake of chemotheraputic drugs into cancerous cells by simultaneous application
of the electric field and drug in the treatment of melanoma [9], Removal of unwanted
cells by localized treatment reduces the level of endangerment to surrounding tissue
compared to more traditional and invasive techniques, such as chemotherapy or surgical
excision. As another example, fusion of two or more cells by joining permeabilized
membrane surfaces have even been developed for the creation of hybrid cells
possessing similar characteristics of the donors [10].
The majority of work in cell manipulation based on field-induced membrane
alterations has focused mostly on the outer membrane, since the generation of the field
required for outer membrane manipulation only is technically easy to develop while
much work remains to understand the phenomenological details of pore formation,
resealing, and molecular transport. Furthermore, a wide range of applications and
additional theory stemming solely from the outer membrane effects will keep
investigators involved at the outer membrane level for years to come. However,
potential lines of inquiry comprise the investigation of field-induced cellular responses
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based on manipulation of membrane structures apart from the plasma membrane which
includes but is not limited to: nuclear membrane, double-walled mitochondrial
membrane, calcium storage vesicles, golgi bodies, endoplasmic reticulum, etc.
Manipulation of each membrane structure arguably has similar, wide-ranging impact in
the medical and biological community since each organelle has a fundamentally
important role which is dependent on the normal, continuous functioning of its
membrane ultrastructure. Thus, the need for investigation at the intracellular level
becomes clear. The major challenge in investigating the intracellular aspects of
membrane perturbations is in developing a method in which it is possible to create the
high intracellular fields necessary to form the transmembrane channel while
simultaneously restricting membrane alterations to a target organelle thus, avoiding the
electroporative effect at the plasma membrane entirely.
Recent lines of study exploring the response of cells to “ultrashort” electric field
pulses with durations of <100 ns and with field strengths as high as 300 kV/cm [11]
provided great inroads into understanding and observing the intracellular effect alone.
Ultrashort pulses are defined as pulses that have a pulse length with durations at full-
width half maximal to be shorter than the capacitive charging time of the outer
membrane at approximately 20 ns. In this shocking regime, nanosecond scale pulsed
electric field (nsPEF) manipulation is different from other manipulative techniques with
the desired effect of inducing morphological changes at the intracellular membrane
structures only [12]. It is theorized that the short pulse duration of nsPEF permits the
intracellular lipid bilayer structures to feel the effects of the large electric field while
avoiding the capacitive charging effect at the plasma membrane thus, avoiding aqueous
pore formation.
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4
This study extends the investigation of field-induced changes in cellular functioning
stemming from membrane perturbations to other lipid bilayer structures not including the
plasma membrane. Since no technology exists to directly monitor the membrane
morphology at the picosecond or nanosecond scale, the experimental goal of this thesis
is to evoke and measure an apoptotic response, with the expectation that sufficient
internal membrane disorganization was inflicted to force the cell to initiate the apoptotic
cascade. Initiation of the apoptotic cascade by application of nsPEF also serves to
validate the feasibility of changing intracellular processes noninvasively while minimizing
the necrotic side-effects. Specific focus is made on eliciting an apoptotic response from
nsPEF electrical insults and on quantifying the degree to which apoptosis can be
induced prior to the observance of other catastrophic events, such as cell necrosis. The
apoptotic response was selected as the process of observation since initiation of
apoptosis based on electromagnetic stimuli has wide ranging implications in cancer
therapy, telecommunications, treatment of heart disease, and the critical role it plays in
the development of an organism. Apoptosis for this study is quantified by measurement
of both the fluorescence emission of one or both dyes relative to the untreated cell
samples and the percentage of cells within a population demonstrating high
fluorescence intensities. Measurements are performed by a flow activated cell sorter
(FACS, Becton-Dickinson). Propidium Iodide and conjugated Annexin V-FITC are the
dyes implemented. Cell viability will be observed by monitoring the percentage of cells
absorbing the trypan blue stain. Additional experiments characterizing the intracellular
effects of nsPEF are performed by observation of the relative cell population
percentages and fluorescence intensities of the dye JC-1 in monomeric or aggregate
form and through microarray gene analysis. Results of the two latter assays are beyond
the scope of this thesis and will not be discussed.
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THEORY-ELECTRICAL
5
Capacitive Membrane Charging
Modeling attempts have been made to further understand the potential field
distribution about the cell due to the dependence of pore formation on the
transmembrane potential. Estimation of the field distribution is based on Laplace’s
Equation for the charge-free system [10, 11]. In this system, several assumptions are
made:
1. The cell is spherical.
2. The membrane is treated as a dielectric material with a relative permittivity of
2, ignoring any resistive property normally associated with the membrane.
3. The extracellular and intracellular space also consists of a dielectric material
with a relative permittivity of 80.
4. Induced pore formation does not have any appreciable effect on the applied
transmembrane voltage.
5. An internal organelle, such as mitochondrion, is spherical.
For both the suspension and the intracellular space,
v2 o= 0 ,
O being the voltage potential of the system with the boundary conditions being
the positive and negative voltages at the cuvette electrodes. A time-dependent solution
to a 2-dimensional system results in the following approximation:
V(t)= 3/2 a E cos 0 (1-exp(-t/T))
0 is defined as the polar angle with respect to the direction of the field, a is the radius of
the cell and t is the charging time constant of the membrane. This closed form solution
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is valid only for an extracellular space with a maximum radius of 3a from the center of
the cell, x, the charging time constant of the system is also characterized by the formula
x = a C ((1/e,) + (1/2ae ))
where < * and oe are the respective intracellular and extracellular conductivities. C is the
capacitance per unit area of the membrane surface. As shown, the magnitude of the
transmembrane voltage V(t) and the time constant changes linearly with respect to the
cell radius, a. Similar conductivities to both the extracellular, intracellular, and vesicular
spaces are assumed. It becomes clear that intracellular organelles, such as
mitochondria, liposomes, and other membrane structures with much smaller radii will
have faster rising times due to the smaller dimensions of a and C. As example to Jurkat-
T lymphocytes and the mitiochondrion within the cell volume (Table 1):
Table 1: Approximate dimensions of Jurkat-T lymphocytes and mitochondrion [12]
Parameter Description Dimension
^ c e ll
Diameter of typical
mammalian cell in suspension
5 pm
3 m ito
Approximate diameter of
mitochondria
.5 pm
r2-ri
Membrane Thickness
(arbitrary)
.005 pm
£ r
Relative Permittivity of
Membrane
2
1 * 1 cell, i* 2 c e ll
Inner membrane cell radius,
Outer membrane cell radius
5 pm , 4.995 pm
l"l m ito , l " 2 m ito
Inner mitochondrion radius,
Outer mitochondion radius
.500 pm, .495 pm
C|. U e
Conductivity 1 S/m, 1 S/m
The spherical capacitance of a dielectric shell is: Cs h e ii = 4 t t er e0 (ri)(r2 ) / (r2 -r1 ),
resulting in (Table 2). From the above equations, it is theoretically possible for the
transmembrane potential of a small intracellular membrane structure to cross the 1 volt
threshold prior to outer membrane perturbations. Applied pulses must be of sufficient
strength to force V(t) beyond 1 volt while keeping the pulse duration itself sufficiently
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short so that the outer membrane is not excessively charged while charging the internal
membrane fully.
Table 2: Derived parameters for capacitive membrane charging ________________________
Parameter Description Dimension
Plasma Membrane
Capacitance
1.1 pF
Mitochondrion Membrane
Capacitance*
11 fF
Ccell
Plasma Membrane Specific
Capacitance
3.50 mF/m^
Cmito
Mitochondrion Outer
Membrane Specific
Capacitance*
3.50 mFlmz
^ cel!
Plasma Membrane Charging
Time Constant
35 ns
T mito
Mitochondrion Charging Time
Constant
3.5 ns
( 5 ) ( t C e||)
Time to Fully Charge the
Plasma Membrane
175 ns
( 5 ) ( t mito)
Time to Fully Charge the
Mitochondrion
17.5 ns
• Mitochondrion capacitance does not represent the inner membrane capacitance
corresponding to the intermembrane space. Calculation of the mitochondrion
capacitance is for the outer membrane only and assuming spherical geometry.
As pren the above example, an ideally square pulse must be much less than 175
ns while the field should be of sufficient strength to induce a 1V transmembrane potential
near 17.5 ns or slightly greater rise times. Field strengths, E, will be on the order of 10’s
of kV/cm. High field generation in combination with short rise times and nanosecond
scale durations place special demands on the power modulator as is discussed
elsewhere.
Lumped Circuit Model
Another model for the electrical response to the applied field is by
characterization of cell components as lumped circuit elements (Figure 1), (Table 3) [13-
16]. The lumped circuit model stems from the work of Schoenbach, where the entire cell
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suspension arrangement is modeled as a capacitor/resistor network driven by a high-
voltage source.
Pulse / - K
Gen. [\J
Rs-
Cs
Cell
Suspension
Intracellular
Structure
Plasma
Membrane
Figure 1: Linear circuit
model of a single cell in
suspension
In modeling the cell, the
simplest approach is to
equate each cell membrane
or aqueous region as a
capacitor or resistor,
respectively. Intracellular
structures are effectively
shielded from external fields
by the plasma membrane.
Although not necessary for
evaluation of current
through the membrane, the
cell suspension Rs, is
sometimes necessary to
evaluate the thermal effects
from power dissipated into
the suspension.
Table 3: Lumped circuit parameters for linear circuit model of a single ce
Circuit Element Dimension
Membrane Capacitance C2 = 2.2 pF, C3 = 22 fF
Aqueous Resistance R2 = 1 MG, R3 = 100 kG
Medium Capacitance &
Resistance
Cs = 14 nF , R3 = 20 Q
The lumped circuit network for an individual cell in suspension can be modeled
as a cascaded RC circuit driven by a voltage source, with the transmembrane potential
voltages being the voltage drop across the equivalent capacitor membranes C2 and C3.
Resistances were obtained as the inverse of the product of the specific conductance and
the approximate organelle size. Capacitances were approximated as double the
computed previous value to equate the result of effective series capacitance. Identical
intracellular specific conductance of the aqueous medium within the arbitrary intracellular
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9
structure and that of the surrounding cytosol are assumed at 1 Siemen/m. The left
portion of the suspension, Rs and Cs can be ignored, since joule heating of the medium
at Rs is negligible. As example to the avoidance of capacitive plasma membrane
shielding, the forcing function to the system will be a monophasic square wave pulse
delivered by the pulse generator with the following parameters (Table 4):
Table 4: Pulse parameters for arbitrary field stimulus to a cell
Parameter Dimension
Rise Time 300 ps*
Pulse Duration at maximum voltage 20 ns
Fall Time 300 ps
Peak Voltage 20 kV
Deriving of a numerical solution using Oread PSPICE yields the following plot (Figure 2)
for the transient voltage drop across the membranes C2 and C3 :
Figure 2: Simulation result of capacitive membrane charging in a
linear circuit model
1.0V
Norrri
VoFta
0.6V
Potential
Normalized
Voltage
0.4V
i . . . . . . . .
Plasma Trans-
Vlembrane Patentiajl 0.2V
0 V
-0.2V
Os 5ns 10ns 15ns 20ns 25ns 30ns
Time fnst
In the lumped circuit model, the membrane potential of an intracellular structure
such as the mitochondria, can reach threshold voltages much more quickly due to the
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10
high-pass filtering effects of the plasma membrane (Fig. 3), which is similar to the
derived membrane effect based on the solution to Laplace’s Equation. Regardless of
the model selected, applied nsPEF is a method capable of eliciting intracellular effects
based on membrane perturbations without inducing the electroporative effect on the
outer membrane. If intracellular processes dependent on membrane partitioning,
especially those of the mitochondria and of the nuclear membrane, are manipulated by
aqueous channel formation resulting from an external electric field, then it becomes
possible to manipulate cellular functions noninvasively by avoidance of plasma
membrane rupturing, or excessive joule heating.
Figure 3: Power spectral density plot of the field stimulus and membrane response
At high
frequencies
greater than 100
MHz, significant
amounts of power
are able to
penetrate through
the plasma
membrane as
shown in the
amplitude of the
sidelobes. The
plasma
membrane does
not experience
much of the field
effect at all since
it’s response lies
mostly within < 50
MHz.
600mV
rane Power Spectral
Applied Pulse Power Spectral Density :
Intracellular Mem orane Power
Spectral Density
0 . 4GHz 0.8G H z
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THEORY - BIOLOGICAL
1 1
Apoptosis
From prior studies, a wide range of observations resulting from E/M interactions
may be induced. Since it was not known precisely which physiologic response will be
elicited from the field exposure, it was decided to focus on an apoptotic response.
Apoptosis is strictly defined in Stedman’s Medical Dictionary as the single deletion of
scattered cells by fragmentation into membrane-bound particles which are phagocytosed
by other cells. It is a genetically mediated form of cell death, characterized by many
biochemical and morphological changes, including cessation of vital cellular processes
and fragmentation of the cell into smaller apoptotic bodies [17] (Figure 4).
Figure 4: Micrograph of an
apoptotic cell, showing typical
fragmented characteristics
Regardless of the inducing agent or
method of cellular injury to evoke
the apoptotic response, all pathways
converge toward the formation of
membrane blebs and apoptotic
bodies with specific biochemical
tags on the outer surfaces which
invite neighboring cells to
phagocytose the smaller
membrane-bound vesicles.
Destruction of cells via the apoptotic
pathway does not evoke an
inflammatory response in
surrounding tissue.
Observation of apoptosis is straightforward, and is directly applicable to
noninvasive tumor suppression. Furthermore, the experimental background for the
detection, quantification, and error in analyzing apoptosis is relatively mature spanning 3
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12
decades. Thus, the need for apoptotic studies becomes clear. References [18] and [19]
provide a detailed review of many of the assays available.
The mitochondrion has been implicated in prior studies as the starting point for
the apoptotic cascade. In particular, an early marker of apoptosis is the collapse of the
mitochondrial membrane potential, possibly by the formation of “permeability transition
pores” which further allow leakage of normally sequestered apoptosis-inducing proteins,
such as Cytochrome C and AIF into the cytosol [20]. It is hypothesized that if the
double-walled membrane structure of the mitochondria is directly breached due to the
field-induced effects, then it is possible for the apoptosis related proteins to diffuse
directly into the cytosol, inducing a forced apoptotic response (Fig. 5) [21].
Figure 5: Mitochondrial membrane rupturing with release of cytotoxic moluecules
Rupturing of the outer mitochondrial membrane results in the cytosolic diffusion of signaling
molecules such as caspases, cytochrome C, or other endogenous molecules strongly linked to
apoptosis, shown in green. The hydrogen ion gradient also collapses and results in the inability
of the mitochondria to convert ADP to ATP in the oxidative phosphorylation process.
Calcium storage vesicles, such as the ER can potentially be manipulated due to
the field-induced effects, possibly resulting in the diffusion of large amounts of Ca2+ into
the cytosol. Similarly, intracellular C a2+ concentration is closely linked to apoptosis
although the precise set of roles within the apoptotic process is not fully determined.
Characterization of Apoptosis-Annexin V Assay
Several methods exist for quantifying the degree of apoptosis in heterogeneous
cell populations since the process of self-regulated cell death contains many observable
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13
pathways with a distinct morphological [19] or biochemical signature for each. A
common method of detecting apoptosis is to observe the translocation of
phosphatidylserine (PS) from the cytoplasmic face of the outer membrane to the
exoplasmic face. It is known that the presence of PS on the outer cell surface provides
a marker for adjacent cells to phagocytose cells expressing the molecule [22], As the
cell progresses through the apoptotic process, PS migrates to the outer leaflet without
any loss of membrane integrity [23], Thus, the mechanism provides a self-regulating
method for neighboring cells to dispose of unwanted tissue immediately.
Optical inspection of PS migration involves tagging the lipid with a fluorescent
dye. Annexin V (AV) is a protein that selectively binds to PS within a certain calcium
concentration. Fluorescein-isothyanate (FITC), a green fluorescent dye, is conjugated to
AV, providing a selective fluorescent marker for PS detection. PS migration is detected
optically through a flow cytometer, as a sharp increase in fluorescence emission of FITC
when excited by the cytometer laser source. Fluorescence intensity characteristic
changes are captured through a series of photomultiplier tubes (PMT). Furthermore, the
angle of light scattered light, which is indicative of the cell morphology, is also captured
by the PMT for qualitative morphological analysis [24]. In evaluating apoptosis in
fractions of the population, it is clear that an apoptotic cell, expressing larger amounts of
PS on the outer surface, would bind with greater amounts of AV-FITC stain, resulting in
an increase in green fluorescence at FITC’s peak wavelength near 530 nm.
Although characterized by biochemical changes such as PS migration or release of
Cytochrome C, the highest standard by which apoptosis is measured, is through the
morphological changes of cell structures since reports exist on various apoptotic
pathways inclusive or exclusive of biochemical markers (i.e., caspase dependent or
independently regulated apoptosis, fragmented or relatively unfragmented DNA @ 300
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14
bp). Changes such as membrane blebbing, chromatin condensation, convolution of the
nuclear envelope, and compartmentalization of the cell are relatively large scale
structural changes to which all apoptotic pathways converge to and is less likely to be
refuted by it’s discrete nature. Thus, apoptosis and necrosis must be further defined
since it is possible to incorrectly diagnose the cellular response and mistake apoptosis
for necrosis or vice versa. Necrosis is defined the pathological destruction of the cell by
severe biochemical or physical insults. This mode of cell death results in rupturing and
lysing of the cell, followed with diffusion of the intracellular solutes which also contribute
to inflammation of surrounding tissue.
In observing apoptosis through an indirect mean such as flow cytometry, it is
important to differentiate the two since both physiologic responses may yield an identical
signal if using a single dye. In late-stage apoptosis and at any time during necrosis, the
outer cell membrane is severely compromised with molecules free to diffuse through the
plasma membrane. Thus, staining with one or any dye is insufficient to differentiate the
mode of death. To further aid in differentiation, a second dye can be simultaneously
introduced to the cell with differing modes of binding and fluorescence. Propidium iodide
(PI) is a common marker for the monitoring of cell necrosis by showing an increase in
Pi’s peak emission at 617 nm when the dye intercalates with DNA. PI is unable to
penetrate into the cell interior in normal cells. However, the dye is free to propagate into
the cytosol when plasma membrane integrity is lost. With two dyes, AV and PI, present
during the cytometric inspection, the following conclusions can be drawn (Table 5):
Table 5: Possible outcomes of the Annexin V / PI assay.
Normal Cells Apoptotic Cells Necrotic or Late
Apoptotic Cells
Annexin V-FITC Faint Green Intense Green Intense Green
Propidium Iodide Faint/No Red Faint/No Red Intense Red
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15
Characterization of Apoptosis - Forward and Side Scatter
The differentiation between normal and apoptotic cells is straightforward with the
observation of a sharp increase in green fluorescence emission, red fluorescence
emission, increase in both, or very little of either. However, the differentiation of late
stage apoptotic vs. necrotic cells is difficult. In the final stages of apoptosis, membrane
blebbing and partitioning are sufficient to reduce the cell size and membrane
composition into manageable vesicles for phagocytosis. However, the vesicles also
become porous, allowing intracellular diffusion of both dyes, making differentiation of
either mode of death difficult. Thus, additional information is required to determine if the
cell response is genetically mediated or not.
The interpretation of both the forward and side scatter profiles of cells serve as another
tool in differentiating between secondary necrosis as a result of apoptosis and necrosis
itself [30] since FSC and SSC changes are governed by the redirection of light through a
combination of diffraction, reflection, and refraction as a function of the cell shape alone
and not by dye uptake. Observations of scatter shifts, based on the apoptotic response,
can be easily observed and is necessary for further identification of apoptotic or necrotic
subpopulations [25-26]. During the cytometric analysis of this study, cells are flowing
uniformly through a small orifice and are illuminated by 488 nm laser light. PMT’s,
located along the beam path and orthogonal to the beam direction, detect the scattered
light and generate a corresponding signal, proportional to the amount of light collected.
To avoid damage to the PMT located in-line of the illuminating beam, a small
obscuration bar is placed to block the majority of light entering. Thus, very little light
reaches the phototube. However, scattered light bent to approximately .5 to 2 degrees
clears the obscuration bar and reach the detector surface. This light incident on the
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16
forward PMT is termed Forward-Angle Light Scatter (FSC). It has been shown that there
is a corresponding decrease in the FSC with the reduction in cell size.
Similarly, PMT’s are located perpendicular to the beam path. If the beam does
not illuminate an object, then very little light should reach the side PMT while viable cells
will always demonstrate a baseline amount of reflected or refracted light entering the
PMT. However, irregularities on the cell surface and the intracellular structure increase
the likelihood of the beam being scattered to one of the side PMT’s [27], Thus, the side
scatter (SSC) can be a measure of the granularity or irregularity of the cell structures. In
the formation of apoptotic bodies and membrane blebs as a result of apoptosis, the cell
surface and its structures become highly irregular and asymmetric. Thus, any shift in the
cell function from normal respiration to apoptosis will result in both a decrease in the
forward angle scatter due to cell shrinkage as well as an increase in the SSC signal due
to the increase in structural irregularities created by the membrane bound vesicles (Fig.
6).
It should be noted that the above morphological changes, as detected by the
FSC or the SSC signal, are also subject to error, as is the case with many biological
assays including that of the Annexin V assay. Thus, the information gathered from
scatter profiles must be coupled with the analysis from other sets of data, including
fluorescence emission shifts, and dye uptake as a function of the entire cell population to
conclude the most likely mode of death [33].
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17
Figure 6: Dot plot migration of cell populations during execution of the apoptotic cascade
analyzed by flow cytometry
m
The dual
staining of
viable cell
samples with
Annexin V
(Panel A)
illustrate
relatively low
fluorescence
emission of
either stain,
since the dyes
do not normally
penetrate the
membrane
boundary.
FSC/SSC plots
show relatively
high forward
scatter profile
with low in the
side scatter
(Panel C).
Apoptotic
samples (Panel
B and D) show
increased
fluorescence
emission along
one quadrant
as the reduction
in forward side
scatter reveals
a reduction in
cell size and
increase in side
scatter typical
of high
granularity in
apoptosis.
A - Control - Fluor,
1 0 *
FL1-Height
C - Control - FSC/SSC
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METHOD
18
Cell Line
For observation of electrically-induced structural changes, development of a
reliable model was necessary for providing consistent and reliable results. The Jurkat-T
cell line was selected for its attractive features. Jurkat cells are spherical cells typically
10 microns in diameter and grow rapidly in physiologic medium with a doubling time
between 24 and 36 hours, which allows easy scheduling and maintenance of cell
cultures. The near spherical shape allows a more precise model for the estimation of
the transmembrane potential (TMP) from prior theoretical studies. Specifically, the intent
at estimating the voltage potential from gross calculations or numerical solutions from
other studies was to determine an upper threshold on rise times and peak voltages to
avoid outer membrane permeabilization. Also necessary was determining if the rise time
and peak voltages of the pulse were sufficient to generate high TMP’s in organelles.
Furthermore, a spherical cell without the added geometrical complexity of processes or
arbors will eliminate the variable on whether any field effect was due to the field
concentration at jagged dielectric interfaces. Jurkat cells are also normally grown in
suspension, as opposed to adhering on the culture vessel itself. Selection of a
suspended cell line removes the need for detachment through cytotoxic enzymes, such
as trypsin, which can potentially skew the data toward an apoptotic response.
Application of an electric field directly on a culture surface is unattractive since the
culture vessel, whether plastic or glass, will most likely have a relative permittivity of
between 2 and 10, which will result in the reduction of the field intensity in the aqueous
region between parallel plates. Finally, prior work from other investigators have set the
stage as Jurkat cells being the most likely cell for nsPEF studies [34, 35]. Studies of a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
19
forced apoptotic response in Jurkat cells using various surfactants allow for a proven
implementation of a positive control data set. Prior studies of electric-field induced
apoptosis into this particular Jurkat cell line give insight as to what type of field
parameters are required to elicit a response since the variability in cell type may result
an equally varied treatment requirement between cell types.
Pulse Generator
The pulse generator used for demonstration of the biological applications of
nsPEF technology is as important as the cell line itself. Commercially available high
voltage pulse generators (PVX-, PVM- series, Directed Energy, Inc.) may not provide the
required pulse characteristics with respect to rise time. Furthermore, these self-
contained generators may require additional auxiliary equipment, such as mating of the
generator to a fluid receptacle. Also, matching of the pulse generator impedance to the
load impedance for optimum pulse characteristics will require tuning and modification of
the generator itself. Thus, it becomes more practical to build a custom system. The
electric field stimulus for this work was applied through a custom nsPEF generator fitted
with an electrode receptacle for holding the cuvette (Dept, of EE-EP, USC) (Figure 7).
Figure 7: Pulse generator hardware used for pulsing of suspended cells
Custom pulse generator constructed by the Dept, of Electrical Engineering at USC (A). The pulse
generator, at lower left, is capable of providing a 2 kV voltage drop across the electrode surfaces
of the cuvette at repetition rates of 20 Hz at a pulse duration of 20 ns. The pulse is delivered by
discharging the pulse forming line through the load of the cell suspension by closure of an
electronically triggered spark gap switch. Flat aluminum plates of the cuvette (B) allow formation
of a uniform electric field within the suspension located in between. The total volume of the
suspension is 75 uL.
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20
Environment
Jurkat-T lymphocytes were cultured in RPMI-1640 growth medium supplemented
with 10% FBS, L-Glutamine, and penicillin/streptomycin antibiotics. Cell suspensions
were cultured at 37°C, 5% C02 and maintained at cell concentrations between 1 x105
and 1 x 106 cells/mL in accordance with American Type Culture Collection (ATCC)
culturing guidelines. To induce apoptosis in cells as a positive control, cells were also
cultured in RPMI-1640 growth medium supplemented, as shown above with an
additional .01% (v/v) Triton X-100 detergent. When culturing cells in detergent laced
medium, cells were allowed to incubate for only up to 5 hours. Cells were centrifuged
and processed for flow cytometric analysis at 0, 2, 5, and 24 hours after shocking. Cells
immersed in detergent supplemented media were only inspected at 2-hour and 5-hour
intervals only since it was shown in prior studies that few cells are intact beyond the 5th
hour.
Cell Suspension Treatment
Jurkat-T lymphocytes, cultured in RPMI-1640 growth medium, were harvested
and resuspended in fresh medium to a final concentration of 2 x 107 cells/mL. 100 uL
aliquots of this suspension were deposited into 1mm electroporation cuvettes and
immediately shocked in accordance with the shocking schedule as shown below.
Shocked cell suspensions were removed from the cuvette, placed in a culture plate, and
incubated until the proper inspection time. Inspection times were at 0, 2, 5, 8, and 24
hours after shocking.
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21
Flow Cytometric Analysis
Aliquots of the Jurkat-T cell suspension were obtained from the culture plate,
centrifuged, and washed in 500 uL of cold PBS. Cells were then centrifuged once more
and resuspended in Ca2+ binding buffer. 5 uL aliquots of PI, and Annexin V-FITC
Table 6: Experiment protocol in tabular format
Shocking schedule and inspection times for cell samples - Cell samples are shocked between 0
and 50 pulses and then placed in cell culturing vessels for continued incubation. As shown, a
small sample is withdrawn from the culture vessel at the selected time interval and analyzed for
cell viability using the Trypan Blue Exclusion or Annexin V assay.
0 Hour 14 Hour 2 Hour 5 Hour 8 Hour 24 Hour 48 Hour
0 pulse AV, TB TB AV, TB AV, TB AV, TB AV, TB TB
2 pulse AV None AV AV AV AV TB
8 pulse AV None AV AV AV AV TB
20 pulse AV None AV AV AV AV TB
50 pulse AV, TB TB AV, TB AV, TB AV, TB AV, TB TB
0 pulse +
TX100
None None AV AV None None None
AV- Annexin V / Propidium Iodide Staining Assay analyzed by flow cytometry
TB - Trypan Blue Exclusion Assay analyzed by direct micrographic inspection
conjugated dyes were deposited into the samples in accordance with manufacturer’s
instructions. After immersion of cells with binding buffer and dyes, cell samples were
allowed to incubate in the dark for 15 minutes and were then analyzed by flow cytometry
(FACStar, Becton-Dickinson) (Figure 8).
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22
B
A
v .
2 kV
c
a a i egg;
.X
*;......
i486 nm Ar
T
/
■*■ '
i486 nm Ar’"
........
*
488 nm Ar
:■ ■ ■ % ...........
488 nm Ar
/
D
Figure 8: Overview of
experiment procedure
(A) Cells are grown in
physiologic medium and allowed
to grow in optimum conditions
prescribed by ATCC. (B) Cells
are harvested and shocked
according to schedule defined
earlier using standard
electroporation cuvettes. (C)
Cells are then deposited into
culture vessels for continued
growth for up to 24 hours. (D)
At various times, cell samples
representative of the various
treatments are withdrawn from
the main batch and assayed by
flow cytometry. Cells are
passed through a small orifice
for individual illumination. The
illuminating source is an Kr/Ar
laser. We are using the 488 nm
wavelength. (E) Upon
illumination, two sets of data are
collected. The first set is the
wavelength emission and
intensity. AV emission at 520
nm reflects PS binding,
indicative of early-stage
apoptosis. PI emission only at
617 nm indicates necrotic cells,
which are dead through a
nonapoptotic pathway. Both
emissions at 617 and 520 nm
suggest either late stage
apoptotic or necrotic cells.
Neither mode of cell death can
be defined precisely due to the
dual staining. Relatively low
emission of both wavelength
suggest normal, viable cells.
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RESULTS
23
Trypan Blue Exclusion
Changes in the estimated number of viable cells are in Table 7.
Table 7: Cell viability count resulting from TBE assay
Control
- 0
pulse
10/5/02
Experiment
- 50 pulse
10/5/02
Population
Change
10/5/02
%
Non-
Viable
Control-
0 pulse
10/17/02
Experiment
- 50 pulse
10/17/02
Population
Change
10/17/02
%
Non-
Viab
le
0
hour
8.26 x
105
2.62x10® -5.64x10® 59% 7.92 x
10®
3.05x10® -4.87x10® 60%
1 /2
hour
7.12 x
105
1.53x10® -5.59x10® 78% 9.72 x
10®
2.76x10® -6.96x10® 67%
1
hour
7.68 x
106
2.96x10® -4.72x10® 56% 9.50 x
10®
3.46x10® 6.04x10® 59%
2
hour
8.12 x
105
7.88x10® -.24x10® 39% 9.72 x
10®
6.86x10® -2.86x10® 7.8%
5
hour
1.07 x
10®
6.68x10® -4.02x10® N/A 1.22 x
10®
9.44x10® -2.76x10® N/A
24
hour
1.88 x
10®
8.85x10® -9.95x10® N/A 2.65 x
10®
1.23x10® -1.42x10® N/A
As shown, all samples show a marked decrease in the number of viable ce Is
remaining within the population after shocking. The percentage of non-viable cells
represents the fraction of the cell population deemed necrotic by uptake of the Trypan
Blue stain. The reduction in cell count in addition to an increase in the non-viable
population demonstrates that the reduction in the remaining viable fraction is not through
attrition in the population alone by overt killing of the cell. Increase in non-viable
percentages show an effect forcing uptake of the dye.
Annexin-FITC / Propidium Iodide Staining
Table 8 and Figure 9 illustrate the increase in Annexin V positive staining where
increased FITC emission at the 512 nm wavelength is the dominant signal.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
24
Table 8: Experiment result: percentage apoptotic yields 10/05/02
20 and 50 pulse treated samples show greatest apoptotic yields. Out of 20 and 50 pulse
samples, a local maxima exists at 2 hours, at which the population shows reduction in the
apoptotic yield, suggesting a recovery from the electrical insult.
0 hours 2 hours 5 hours 24 hours
0 pulse 4 8 6 4
2 pulse 5 7 7 3
8 pulse 6 8 6 5
20 pulse 19 35 19 7
50 pulse 45 45 41 1 1
Trition X-
100
5 37 47 N/A
Figure 9: Experiment result: percentage apoptotic yield 10/05/02
Percentage Apoptotic Yield
10/5/02
Ann^xifT
Posftivi!
Fracticr
(%)
Pulse
20
Pulse
-©■■Triton X -
100 Pulse
0 5 1 1 2 2
(hours)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25
Table 9 and Figure 10 illustrate similar increases for the experiment on 10/17/01.
Table 9: Experiment result: percentage apoptotic yields for 10/17/01
Gated Percentages for Annexin V positive, apoptotic fractions on the experiment of 10/17/02.
Results also show 20 and 50 pulse treated samples showing greatest apoptotic yields at 2 hours
with decrease in yields afterwards, suggesting a recovery from the electrical insult.
0 hours 2 hours 5 hours 24 hours
0 pulse 3 4 6 3
2 pulse 2 6 6 3
8 pulse 5 1 1 12 4
20 pulse 26 42 37 9
50 pulse 53 62 53 12
Trition X-100 5 37 47 N/A
Figure 10: Experiment result: percentage apoptotic yield 10/17/01
Percentage Apoptotic Yield
10/17/02
Annexin V
Positive
Fraction (%)
— 0 Pulse 2 Pulse
-^— 8 Pulse
-B-5C Pulse Triton X-100
0 5 10 15 20 25
Time (hours)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION
26
Results demonstrate the ability for nsPEF to elicit cellular responses affecting
cellular functioning beyond that of only localized pore formation, regardless of the
membrane surface of interest. nsPEF stimulus induces changes in cellular functioning
as severe as forced initiation of the apoptotic cascade, since the reduction in cell viability
reported by TBE in addtion to the fluorescence changes by AV-FITC binding to PS
implies this. It is likely that the apoptotic response from the electric field insult results
from the intracellular membranes's inability to partition and isolate molecular species
normally compartmentalized in the cell. Loss of membrane integrity, allowing the
diffusion of normally sequestered molecules, can be further linked to either the
disorganization of the phospholipid bilayer structure or the formation of aqueous pores
spanning the entire width of the membrane cross-section. Prior studies from various
investigators implicate several molecular or ionic species requiring compartmentalization
for viable homeostasis. Cytochrome C, and Apoptosis Inducing Factor (AIF) are two
such molecules normally isolated within the mitochondria. Reorganization of the double
walled membrane structure resulting in release in either species increases the likelihood
of entering the apoptotic cascade. Sufficient membrane perturbation can result in
sphingomyelin (SM) translocation from the exoplasmic face to the cytoplasmic face,
where it is then hydrolyzed to Ceramide. This conversion process, in combination with
PS reorganization, leads to vesicle shedding and membrane bleb formation which are
also apoptotic characteristics of a cell. Any PS reorganization effected by the electric
field may be matched with a similar effect on SM translocation. Other possibilities exist
on why the apoptotic cascade is initiated shortly after the onset of high electric fields
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
27
since apoptosis can be triggered by many pathways, several of which are not listed here.
Investigation into the precise interaction of the electric field modifying cellular functioning
is left to future work.
Since apoptotic cell fractions reached maximal levels at about two hours with an
increase in cell viability afterwards, the declining changes in the apoptotic fraction also
suggest a reversible mode of AV binding to PS. Apoptosis detected by the Annexin
assay is irreversible with respect to the detection method since biochemical events
triggering the apoptotic pathways such as cleavage of the Caspase family of proteins
must precede the phospolipid inversion mechanism. Thus, the decline in AV binding
after two hours suggest an electric-field mediated process of repositioning PS to the
outer leaflet. An electroporative effect allowing a similar result is not likely since an EP
response would have resulted in a dual staining of both increased green and red
emissions. The majority of dual staining yields was relatively low at 21 % or less for both
experiments. Similarly, single staining yields for PI only were less than 11%, which is
typical for cells in the control population.
An alternative possiblity for the unexpected decrease in AV binding is the
phagocytic activity of neighboring cells. The typical lifetime of an apoptotic cell in situ is
relatively short, with durations shorter than the time required for mitosis. The rapid
disappearance of an apoptotic cell results from the phagocytic activity of its neighbors.
In suspensions or tissue culture, the opportunity for intercellular activity is much lower,
thus prolonging the sequestration rate. Futhermore, phagocytic activity may not even be
possible, with disintegration of the dying cell into fragmented bodies, which may also
lower the apoptotic yield as reported by the flow cytometer.
Initiation of apoptosis through membrane disorganization or through other
intracellular means as a result of applied external fields has been successfully validated
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28
through this study. Further development of this technology will allow possible advances
in the noninvasive deletion of undesired tissue or the deliberate manipulation of other
intracellular processes to evoke a desired response.
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29
CONCLUSION
Validation of ultrashort pulses to elicit responses as decisive as apoptosis
compels inquiry into the specificity of the response and the types of structures or cellular
mechanisms it can perturb. For example, it is not known in the current study if apoptosis
is a result of a single perturbed phenomena, such as membrane reorganization or a
wide-sense response in which several processes are negatively affected to collectively
force the cell into the cascade. The various processes of interest will demand additional
methods for precise monitoring. Field effects on microscopically viewable structures will
require new optical technologies currently in development. For example, observation
and characterization of cellular changes or events in the picosecond and nanosecond
regime may necessitate a time-resolved approach towards optical microscopy. Methods
of high-field pulse delivery for various applications involve development of new pulsed
power technology for proper delivery of energy to the load with high peak power over
extremely short duration.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
30
References
[1] Benz, R., Zimmerman, U. (1979). Pulse-Length Dependence of the Electrical
Breakdown in Lipid Bilayer Membranes, Biochimica Et Biophvsica Acta,
597(3):637-42.
[2] Neumann, E., Sowers, A., Jordan, C. Electroporation and Electrofusion in
Cell Biology, 1s t ed. New York, Plenum Press, 1989.
[3] Weaver, J., Chizmadzhev, Y. (1996). Theory of Electroporation: A Review,
Bioelectrochemistrv and Bioenerqetics, 41: 135-60.
[4] Zimmerman, U., Benz, R. (1980). Dependence of the Electrical Breakdown
Voltage on the Charging Time in Valonia Utricularis, Journal of Membrane
Biology. 53: 33-43.
[5] Abidor, I., et al. (1979). Electric Breakdown of Bilayer Lipid Membranes I:
The Main Experimental Facts and Their Qualitative Discussion,
Bioelectrochemistrv and Bioenerqetics. 6: 37-52.
[6] Chang, D., Reese, T. (1990). Changes in Membrane Structure Induced by
Electroporation as Revealed by Rapid-Freezing Electron Microscopy,
Biophysical Journal. 58(1): 1-12.
[7] Dev, S., et al. (2000). Medical Applications of Electroporation. IEEE
Transactions on Plasma Science. 28(1): 206-23.
[8] Zimmerman, U., Vienken, J., Pilwat, G., (1980). Development of Drug Carrier
Systems: Electrical Field Induced Effects in Cell Membranes,
Bioelectrochemistrv and Bioenerqetics. 7: 553-74.
[9] Hoffman, et al. (1999). Electroporation Therapy: A New Approach for the
Treatment of Head and Neck Cancer, 46(6): 752-59.
[10] Zimmerman, U., Neil, G. Electromanipulation of Cells. 1s t ed., New York:
CRC Press, 1996.
[11] Bilska, A., DeBruin, K., Krassowska, W. (2000). Theoretical Modeling of the
Effects of Shock Duration, Frequency, and Strength on the Degree of
Electroporation, Bioelectrochemistrv. 51(2): 133-43.
[12] Foster, K. (2000). Thermal and Nonthermal Mechanisms of Interaction of
Radio-Frequency Energy with Biological Systems, IEEE Transactions on
Plasma Science. 28(1): 15-23.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
31
[13] Schoenbach, K., Joshi, R., Stark, R. (2000). Bacterial Decontamination of
Liquids with Pulsed Electric Fields, IEEE Transactions on Dielectrics and
Electrical Insulation. 7(5): 637-45.
[14] Schoenbach, K. Beebe, S., Buescher, S. (2001). Intracellular Effect of
Ultrashort Electrical Pulses, Bioelectromaqnetics. 22(6): 440-8.
[15] Schoenbach, K., et al. (1997). The Effect of Pulsed Electric Fields on
Biological Cells: Experiments and Applications, 25(2): 284-92.
[16] Schoenbach, et al. (2000). Electromagnetic Effects on Biological Cells, 25th
International Conference on Infared and Millimeter Waves.
[17] Kerr, J., Wyllie, A., Currie, A. (1972). Apoptosis: A Basic Biological
Phenomenon With Wide Ranging Implications in Tissue Kinetics, British
Journal of Cancer. 26(4): 239-57.
[18] McKenna, S., McGowan, A., Cotter, T., Molecular Mechanism of
Programmed Cell Death, in Apoptosis. Al-Rubeai, M., Editor, Berlin:
Springer-Verlag, pp. 1-31, 1998.
[19] Darzynkiewicz, Z., Measurement of Apoptosis, in Apoptosis. Al-Rubeai, M.,
Editor, Berlin: Springer-Verlag, pp. 33-73,1998.
[20] Takeyama, N., Miki, S., Hirakawa, A., Tanaka, T. (2002). Role of the
Mitochondrial Permeability Transition and Cytochrome C Release in
Hydrogen Peroxide-Induced Apoptosis, Experimental Cell Research. 274(1):
16-24.
[21] Wang, X. (2001). The Expanding Role of Mitochondria in Apoptosis. Genes
and Development. 15(22): 2922-33.
[22] Martin S. (1995). Early Redistribution of Plasma Membrane
Phosphatidylserine is a General Feature of Apoptosis Regardless of the
Initiating Stimulus: Inhibition by Overexpression of Bcl-2 and Abl, Journal of
Experimental Medicine. 182(5): 1545-56.
[23] Van Engeland, M., Nieland, L., Ramaekers, F., Schutte, B., Reutelingsperger,
C. (1998). Annexin V-Affinity Assay: A Review on an Apoptosis Detection
System Based on Phosphatidylserine Exposure, Cytometry. 31(1): 1-9.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
32
[24] Benson, M., McDougal, D., Coffey, D. (1984). The Application of
Perpendicular and Forward Light Scatter to Assess Nuclear and Cellular
Morphology, Cytometry. 5(5): 515-22.
[25] Dive, C., Gregory, C., Phipps, D., Evans, D., Milner, A., Wyllie, A. (1992).
Analysis and Discrimination of Necrosis and Apoptosis (Programmed Cell
Death) by Multiparameter Flow Cytometry, Biochimica Et Biophvsica Acta.
1133(3): 275-85.
[26] Ormerod, M., Paul, F., Cheetham, M., Sun, X. (1995). Discrimination of
Apoptotic Thymocytes by Forward Light Scatter, Cytometry, 21(3): 300-4.
[27] Kerker, M. (1983). Elastic and Inelastic Light Scattering in Flow Cytometry,
Cytometry. 4(1): 1-10.
[28] Darzynkiewicz, Z. (2001). Difficulties and Pitfalls in Analysis of Apoptosis,
Methods in Cell Biology. New York: Academic Press, pp. 527-46.
[29] Hofmann, F., Ohnimus, H., Scheller, Cl, Strupp, W., Zimmerman, U., Jassoy,
C. (1999). Electric Field Pulses can Induce Apoptosis, Journal of Membrane
Biology. 169(2): 103-9.
[30] Strupp, W., et al. (2000). Treatment of Cells with Detergent Activates
Caspases and Induces Apoptotic Cell Death, Journal of Membrane Biology.
175(3): 181-9.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
33
REFERENCES (Alphabetical)
Abidor, I., et al. (1979). Electric Breakdown of Bilayer Lipid Membranes I: The Main
Experimental Facts and Their Qualitative Discussion, Bioelectrochemistrv and
Bioenerqetics. 6: 37-52.
Benson, M., McDougal, D., Coffey, D. (1984). The Application of Perpendicular and
Forward Light Scatter to Assess Nuclear and Cellular Morphology, Cytometry. 5(5):
515-22.
Benz, R., Zimmerman, U. (1979). Pulse-Length Dependence of the Electrical
Breakdown in Lipid Bilayer Membranes, Biochimica Et Biophvsica Acta. 597(3):637-
42.
Bilska, A., DeBruin, K., Krassowska, W. (2000). Theoretical Modeling of the Effects
of Shock Duration, Frequency, and Strength on the Degree of Electroporation,
Bioelectrochemistrv. 51(2): 133-43.
Chang, D., Reese, T. (1990). Changes in Membrane Structure Induced by
Electroporation as Revealed by Rapid-Freezing Electron Microscopy, Biophysical
Journal. 58(1): 1-12.
Darzynkiewicz, Z., Measurement of Apoptosis, in Apoptosis. Al-Rubeai, M., Editor,
Berlin: Springer-Verlag, pp. 33-73, 1998.
Darzynkiewicz, Z. (2001). Difficulties and Pitfalls in Analysis of Apoptosis, Methods
in Cell Biology. New York: Academic Press, pp. 527-46.
Dev, S., et al. (2000). Medical Applications of Electroporation, IEEE Transactions on
Plasma Science. 28(1): 206-23.
Dive, C., Gregory, C., Phipps, D., Evans, D., Milner, A., Wyllie, A. (1992). Analysis
and Discrimination of Necrosis and Apoptosis (Programmed Cell Death) by
Multiparameter Flow Cytometry, Biochimica Et Biophvsica Acta. 1133(3): 275-85.
Foster, K. (2000). Thermal and Nonthermal Mechanisms of Interaction of Radio-
Frequency Energy with Biological Systems, IEEE Transactions on Plasma Science.
28(1): 15-23.
Hoffman, et al. (1999). Electroporation Therapy: A New Approach for the Treatment
of Head and Neck Cancer, 46(6): 752-59.
Hofmann, F., Ohnimus, H., Scheller, Cl, Strupp, W., Zimmerman, U., Jassoy, C.
(1999). Electric Field Pulses can Induce Apoptosis, Journal of Membrane Biology,
169(2): 103-9.
Kerker, M. (1983). Elastic and Inelastic Light Scattering in Flow Cytometry,
Cytometry. 4(1): 1-10.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
34
Kerr, J., Wyllie, A., Currie, A. (1972). Apoptosis: A Basic Biological Phenomenon
With Wide Ranging Implications in Tissue Kinetics, British Journal of Cancer. 26(4):
239-57.
Martin S. (1995). Early Redistribution of Plasma Membrane Phosphatidylserine is a
General Feature of Apoptosis Regardless of the Initiating Stimulus: Inhibition by
Overexpression of Bcl-2 and Abl, Journal of Experimental Medicine. 182(5): 1545-
56.
McKenna, S., McGowan, A., Cotter, T., Molecular Mechanism of Programmed Cell
Death, in Apoptosis. Al-Rubeai. M., Editor, Berlin: Springer-Verlag, pp. 1-31, 1998.
Neumann, E., Sowers, A., Jordan, C. Electroporation and Electrofusion in Cell
Biology. 1s t ed. New York, Plenum Press, 1989.
Ormerod, M., Paul, F., Cheetham, M., Sun, X. (1995). Discrimination of Apoptotic
Thymocytes by Forward Light Scatter, Cytometry, 21(3): 300-4.
Schoenbach, K., et al. (1997). The Effect of Pulsed Electric Fields on Biological
Cells: Experiments and Applications, 25(2): 284-92.
Schoenbach, K. Beebe, S., Buescher, S. (2001). Intracellular Effect of Ultrashort
Electrical Pulses, Bioelectromaanetics. 22(6): 440-8.
Schoenbach, et al. (2000). Electromagnetic Effects on Biological Cells, 25th
International Conference on Infared and Millimeter Waves.
Schoenbach, K., Joshi, R., Stark, R. (2000). Bacterial Decontamination of Liquids
with Pulsed Electric Fields, IEEE Transactions on Dielectrics and Electrical
Insulation. 7(5): 637-45.
Strupp, W., et al. (2000). Treatment of Cells with Detergent Activates Caspases and
Induces Apoptotic Cell Death, Journal of Membrane Biology. 175(3): 181-9.
Takeyama, N., Miki, S., Hirakawa, A., Tanaka, T. (2002). Role of the Mitochondrial
Permeability Transition and Cytochrome C Release in Hydrogen Peroxide-Induced
Apoptosis, Experimental Cell Research. 274(1): 16-24.
Van Engeland, M., Nieland, L., Ramaekers, F., Schutte, B., Reutelingsperger, C.
(1998). Annexin V-Affinity Assay: A Review on an Apoptosis Detection System
Based on Phosphatidylserine Exposure, Cytometry. 31(1): 1-9.
Wang, X. (2001). The Expanding Role of Mitochondria in Apoptosis, Genes and
Development. 15(22): 2922-33.
Weaver, J., Chizmadzhev, Y. (1996). Theory of Electroporation: A Review,
Bioelectrochemistrv and Bioenergetics. 41: 135-60.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
35
Zimmerman, U., Benz, R. (1980). Dependence of the Electrical Breakdown Voltage
on the Charging Time in Valonia Utricularis, Journal of Membrane Biology. 53: 33-43.
Zimmerman, U., Neil, G. Electromanipulation of Cells, 1s t ed., New York: CRC
Press, 1996.
Zimmerman, U., Vienken, J., Pilwat, G., (1980). Development of Drug Carrier
Systems: Electrical Field Induced Effects in Cell Membranes, Bioelectrochemistrv
and Bioenerqetics, 7: 553-74.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Young, Clayton Ming (author)

Core Title Initiation of apoptosis by application of high-intensity electric fields

School Graduate School

Degree Master of Science

Degree Program Biomedical Engineering

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,engineering, biomedical,engineering, electronics and electrical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-302313

Unique identifier UC11341162

Identifier 1414893.pdf (filename),usctheses-c16-302313 (legacy record id)

Legacy Identifier 1414893.pdf

Dmrecord 302313

Document Type Thesis

Rights Young, Clayton Ming

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA