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Protein nitrosylation

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Title:
Protein nitrosylation potential mechanism of resveratrol synergy with radiation in cancer treatment
Creator:
Igolnikov, Ilya
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Language:
English
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xiii, 56 leaves : ; 28 cm

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Subjects / Keywords:
Radiotherapy ( lcsh )
Resveratrol ( lcsh )
Radiotherapy ( fast )
Resveratrol ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 50-56).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Ilya Igolnikov.

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|University of Colorado Denver
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|Auraria Library
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ocn519488752
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PROTEIN NITROSYLATION: POTENTIAL MECHANISM OF RESVERATROL
SYNERGY WITH RADIATION IN CANCER TREATMENT
By
Ilya Igolnikov
B.A., University of Colorado Boulder, 2007
A thesis submitted to the
University of Colorado Denver
In partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2009


This thesis for the Master of Science
degree by
Ilya Igolnikov
has been approved
by
Barbara A Frederick
Mark Anderson
zoo9!
Date


Igolnikov, Ilya (M.S., Chemistry)
Protein nitrosylation: Potential Mechanism of Resveratrol Synergy with Radiation in
Cancer Treatment.
Thesis directed by professor Douglas Dyckes
ABSTRACT
Cancer is quickly becoming one of the most common causes of death both in
the US and around the world. Over a million people are diagnosed with cancer every
year and one third of those diagnosed dont survive ten years from that time. Despite
advances in surgical technique and cheniotherapeutics, the standard of care for
surgically inoperable tumors remains to be radiation therapy. Because of genetic
mutations in cancer such as loss of function p53 mutations, radiation is not only an
ineffective apoptotic inducing agent; it also has been shown to enhance metastatic
potential of tumors. Resveratrol, a polyphenol found in numerous plant sources, has
shown to possess chemotherapeutic, weight loss, and age longevity properties among
others. It has been shown that antioxidants such as resveratrol synergize with
radiation treatment, but no verdict on resveratrols synergism with radiation exists.
Furthermore the mechanism of action for resveratrol synergy with radiation is
currently unclear. Through several growth assays, synergy is demonstrated between
resveratrol and radiation. Utilizing a relatively novel methodology called the Biotin


Switch Technique in which nitrosyl groups on cysteines are replaced with biotin
markers and separated from all other proteins, it is also demonstrated that cell-wide
and specific protein nitrosylation is correlated with the observed side-effects of
radiation treatment. The hypothesis that reversal of nitrosylation is at least partially
responsible for synergy between resveratrol and radiation may be supported by this
observation.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed:
Douglas Dyckes


DEDICATION
I dedicate this thesis to my parents, who gave me an appreciation of learning and
taught me the value of perseverance and resolve. They took a huge risk coming to this
country 21 years ago for which I can only hope to someday repay them.


ACKNOWLEDGEMENT
I would like to thank my advisor, Barbara A Frederick for putting up with me in her
lab. For answering questions no matter how mundane, for heckling my scrupulous
washing, for her insight because... clearly she knows everything and lastly for her
unquestioning support when others turned me away.


TABLE OF CONTENTS
Figures........................................................................xi
Tables........................................................................xii
Equations....................................................................xiii
Chapter
1. Introduction...............................................................1
1.1 Cancer.....................................................................1
1.1.1 Significance/Treatment Options............................................1
1.1.2 Limitations of Radiation Therapy..........................................2
1.2 Protein Nitrosylation.....................................................3
1.2.1 Protein Signaling.........................................................3
1.2.2 iNOS......................................................................4
1.2.3 Oxidative Phosphorylation.................................................4
1.2.4 Intentional Mitochondrial Release.........................................5
1.2.5 Comparison to Phosphorylation.............................................5
1.2.6 Method of Detection.......................................................6
1.2.7 Affect of Treatment on Nitrosylation......................................7
vii


1.3 Resveratrol
8
1.3.1 Warburg Hypothesis........................................................8
1.3.2 Oxidative Balance in Cancer...............................................9
1.3.3 Resveratrols Systemic Effects............................................9
1.3.4 Resveratrols Known Actions...............................................9
1.3.5 Why Resveratrol..........................................................10
1.4 Reasoning for Combining Radiation and Resveratrol.........................10
1.4.1 Specificity Mitochondrial Action.........................................10
1.4.2 Effects Beyond Single Pathway............................................10
1.4.3Similarities Between Resveratrol and Radiation............................11
1.5 Purpose of Thesis.........................................................12
1.5.1 Hypothesis...............................................................12
1.5.2 Specific Aim 1..........................................................12
1.5.2 Specific Aim 2..........................................................12
1.5.3 Specific Aim 3..........................................................13
1.6 Scope of Thesis......................................................... 13
1.6.1 Limitations of data......................................................14
1.7 Arrangement of Thesis.....................................................15
viii
2. Methods
16


2.1 Materials..................................................................16
2.2 Cell Culture...............................................................17
2.3 Resveratrol/Radiation Treatment............................................17
2.3.1 Generic Procedure.........................................................17
2.3.2 Specific Protocol Details................................................18
2.4 MTT/Inhibition Assay.......................................................18
2.5 Clonogenic/Proliferation Assay.............................................19
2.6 Protein Assay..............................................................21
2.7 Biotin Switch Nitrosylation................................................21
2.8 Isoelectric Focusing..................................................... 23
2.9 SDS-Polyacrylamide Gel-Electrophoresis (PAGE)..............................23
2.10 Silver Staining...........................................................25
2.11 Mass Spectrometry.........................................................26
3. Results/Discussion..........................................................27
3.1 Clonogenic/Inhibition Assays...............................................27
3.1.1 Multiple Synergy Assays................................................. 31
3.1.2 MTT Deficiences..........................................................31
3.1.3 Clonogenic Deficiences...................................................32
3.1.4Effects of DNA Damage......................................................33
IX


3.1.5 EMT.....................................................................34
3.2 Biotin Switch/Gel Electrophoresis/Silver Staining........................37
3.2.1 Challenges..............................................................38
3.2.2 Experimental Setup....................................................... 39
3.2.3 Alternative Setup.......................................................39
3.3 Mass Spectrometry.......................................................42
4. Conclusion.................................................................45
4.1 Resolution of Hypothesis..................................................45
4.2 Future Directions........................................................45
Bibliography..................................................................49
i i
x


LIST OF FIGURES
Figure
1.1 Known Mechanisms of Radiation Therapy on Tumor Cells....................1
1.2 Physiologic Phosphate Donor ATP.........................................3
1.3 Physiologic Nitrosyl Donor GSNO.........................................4
1.4 Simplified Description of Biotin Switch Technique.......................6
1.5 Protein Nitrosylation Mechanism.........................................7
1.6 Resveratrol Structure...................................................8
1.7 Oxidative Stress Mechanism..............................................8
1.8 Resveratrols Known Direct Interaction and Mechanism....................9
1.9 Proteins Affected By Resveratrol...................................... 11
2.1 CysNO Spectroscopic Reading............................................22
3.1 MTT Cl Values........................................................ 28
3.2Clonogenic Cl Values....................................................29
3.3 Pictoral Representation of Clonogenics.................................33
3.4 Examples of Clonogenic Data Drawbacks.................................34
3.5 Different Appearance of Cancer Cell DNA...............................35
3.6 Comparing Biotin Switch and S-FLOS....................................40
3.7 SDS-PAGE Data.........................................................42
XI


LIST OF TABLES
3.1 Protein band identification
44

t
Xlt


LIST OF EQUATIONS
2.1 Dose-Effect Relationship................................................19
2.2 Confidence Interval.....................................................20
xiii


1. Introduction
1.1 Cancer
Almost 15 million people within the U.S. have been diagnosed with cancer
over the past decade (Jemal et al., 2008) and over one-third of those diagnosed have
died from the disease over the same time period. (Jemal et al., 2008) Treatment
usually options consist of a combination of approaches, termed primary therapy for
the primary approach and adjuvant therapy for the secondary approach. Primary
therapy typically consists of removing the known tumor sites, whereas adjuvant
therapy focuses on chemically aiding in the apoptotic pathways specific to tumor
cells, preventing metastatic growths as well as preventing future tumor recurrence.
Over the past 25 years, advancements in tumor biology have promised a tailored
therapy by developing molecular biomarkers that predict patient response to therapy.
However, surgical removal of tumors remains the preferential method of primary
treatment in most cases. Irradiation is the standard of care for many tumors in
unreachable locations such as head and neck tumors. (Ringborg et al., 2001) The
desired effect of ionizing radiation (IR) therapy are DNA damaging double strand
breaks and cell cycle arrest, (Li et al., 2001) which inhibit cellular growth and cause
damage to DNA such that the cell activates apoptosis and necrosis pathways.
(Shimamura et al., 2005) Assuming regiospecificity of treatment as well as intact
apoptotic mechanisms in the tumor, this would seem to be an ideal method of
controlling disease for tumors that are inaccessible to surgery.
l


Figure 1.1 Known Mechanisms of Radiation Therapy on Tumor Cells. Taken from Li et al., 2001
Unfortunately, one of the most important pathways in radiation induced
apoptosis (El-Deiry et al., 2003) does not function properly in over 60% of tumors.
(Soussi et al., 2001) p53 is a key mediator of tumor suppression and its expression is
altered by deletions, mutations, or loss of homozygosity events that affect the p53
pathway. Radiation affects various cell functions in addition to its effect on DNA.
Proteomic analysis suggest that more than one hundred proteins are affected by
ionizing radiation (Miller Jenkins et al., 2008) with functional implications that range
from regulation of vascular tone, platelet aggregation, neurotransmission, cellular
apoptosis, and immune activation. (Stuehr et al., 1992; Moncada et al., 1991; Ignarro,
1991; Bredt et al., 1990; Hibbs et al., 1990; Lane, Gross 1999) These off-target
effects of radiation have several implications including resistance to
2


apoptotic/necrotic cell death as well as increased aggressiveness and frequency in
later recurrence.
1.2 Protein Nitrosylation
Proteins can signal to each other in a multitude of ways. The three
dimensional structure of proteins allows them to dock onto one another to form
complexes. These complexes either signal or block a signal from proceeding down a
prototypical post-translational modification is phosphorylation, a modulator of
metabolic pathways. Physiologically adenosine triphosphate (ATP) is the basic
phosphate receptor of choice in the body, which can donate a phosphate to other
proteins or enzymes in need of a phosphate and in the process becomes adenosine
diphosphate (ADP). Nitric oxide is believed to have a similar systemic donor
molecule in the form of S-nitrosylated glutathione with which proteins can be
reversibly nitrosylated.
Among these proteins affected by radiation, a subset is targeted for the post
translational modification of nitrosylation. The initially proposed mechanism of
Fig. 1.2 Example of Canonical Phophate Donor/Receiver
Molecule Pair Adenosine Triphosphate (ATP)/Adenosine
Diphosphate (ADP). Taken from Essential Cell Biology,
Garland Science, 2009.
phosphoanhydride bonds
chain of proteins that
together make up a
pathway that serves a
specific function. The
three-dimensional structure
of the proteins is modified
by post-translational
modifications on certain
residues that allows for or
inhibits the interactions of
multiple proteins. The
3


protein
nitrosylation was
thought to be
mediated by IR
inducible nitric
oxide synthase
(iNOS) activation,
(Freeman et al
2000) a protein
that acts to
increase
intracellular nitric oxide production and which has been shown to have an effect on
intracellular protein nitrosylation levels. Due to the similarity in structure/function of
iNOS to other members of the nitric oxide synthase (NOS) family of proteins, the
relationship of endothelial nitric oxide synthase (eNOS) to radiation induced
nitrosylation has also been investigated in various cell types. (Weller et al., 2003)
In addition to NOS producing nitric oxide, several other mechanisms of nitric
oxide production have been reported as well. Nitric oxide and other reactive nitrogen
and oxygen species are incorporated into the oxidative phosphorylation process of the
electron transport chain in the mitochondria. When this pathway is functioning
properly, molecular oxygen is converted to water through several radical
intermediates, resulting in the production of large quantities of energy in the form of
ATP. However, when the protein complex in the mitochondrial membrane becomes
uncoupled by way of deletion or malfunction of the mitochondrial complex proteins,
the radical intermediates may be erroneously released from the mitochondria. The
free nitrosyl groups are initially quenched by cellular antioxidant mechanisms, but if
ASIAS
Fig. 1.3 Similar to ATPs role for Phosphate, Nitrosylated Glutathione
(GSNO) is Considered the Most Physiologically Relevant NO Donor,
Shown Here at the Protein and Biochemical Levels. Taken from Mitchell
et al., 2005 and Hess et al., 2005.
4


the radicals exceed the antioxidant capacity, they can be free to interact with proteins
or nucleic acids.
Free radicals are also sometimes intentionally released from the mitochondria.
The function of this radical release has traditionally been thought to be a dump of
damaging agents into the cytoplasm during apoptosis, but recently it has been
proposed that this mitochondrial originating radical dump could be signaling through
protein nitrosylation. (Verma et al., 2007, Chatterjee et al., 2006)
Proteins are nitrosylated in a similar manner to that in which they are
phosphorylated. Phosphorylation can take place at either serine, tyrosine or threonine
amino acid residues in a reversible manner by binding to the side-chain terminal
hydroxyl group of any of the three aforementioned amino acids. Enzymes known as
kinases and phosphatases have been discovered that facilitate addition and removal of
phosphate groups from various proteins. The purpose of this post-translational
modification is to change the three-dimensional conformation of the protein, either
through changes in the three-dimensional structure of the protein revealing otherwise
hidden amino acids that can interact with other proteins or by changing the site of
phosphate binding itself such that other docking proteins can bind with greater or less
affinity.
Similarly, nitrosyl groups have been shown to directly bind to proteins at their
sulfur containing cysteine residues and alter the functionality of their signaling
pathways (Stamler et al., 1998) and the mediators S-glutathione and cysteine act as an
accessory enzymes to facilitate addition and removal of nitrosyl groups. (Goldstein et
al., 1996; Hess et al., 2005) All these similarities in action would suggest that
nitrosylation acts as an alternative to phosphorylation in facilitating intracellular
signaling pathways. Although post-translational modification can occur via oxidation,
nitration, or nitrosylation; nitrosylation has been implicated as the most
5


physiologically significant modification, even being mentioned as potentially the
next phosphorylation (Mannick et al., 2002) based on its potential significance in
cell signaling. It has been proposed that nitrosylating proteins could serve as a quick
reversible signal to warn of intracellular damage or more specifically cellular
oxidative stress.
o
ll
-S-NO
Protein nitrosylation is clearly an area of research in need of exploration, and
new methods have been created to detect and analyze protein nitrosylation. Ideally, a
method would allow for nitrosylated proteins to be separated from remaining proteins
of the cell and identified in a shotgun approach. The most notable of these methods is
that of Jaffey et al. termed The Biotin Switch. (Jaffrey et al., 2001) In this
procedure, S-nitroso-glutathione
-S-S-CH3 (GSNO) or S-nitroso-cysteine
(CysNO) is added to cell lysates.
This is followed by blocking free
thiols by incubation with thiol-
specific methyltiolating agent
methyl methanethiosulfonate
(MMTS). Nitrosylated thiols are
then reduced to thiols with ascorbate
and the newly formed free thiols are
reacted with Biotin-HPDP, a
sulphydryl-specific biotinylating
reagent. This procedure replaces
SH CH3-S-S-CH3 II 0
S-NO MMTS
Step 1
S*j
1. MMTS removed by I
spin column or acetone gtep 2
2. Ascorbate
"'S-S-biotin
Biotin-HPDP
Step 3
Figure 1.4 Simplified Description of Biotin Switch
Technique. Taken from Jaffrey et al., 2001.
nitrothiol modifications with stable disulfide bonds attached to biotin markers, in
effect allowing one to detect and isolate nitrosylated proteins, a technique that has
been under development for over two decades. (Stuchbury et al., 1975) When
combined with gel electrophoresis and mass spectrometry, this technique allows for
6


the identification of nitrosylated proteins. GSNO was chosen because it is thought of
as the most common donor molecule for cellular transnitrosylations in vivo, often
referred to as a nitrosylation pool within the cell. However the reaction rate of GSNO
is very slow and some experimenters have switched to using CysNO because of its
faster rate of NO exchange with proteins while maintaining specificity and
mechanism similar to that of GSNO.
By utilizing the biotin switch assay, it is possible to observe how various
treatments affect protein nitrosylation levels. Several groups have shown that
different proteins possess specific nitrosylatable cysteine sites and that these are
physiologically relevant to their function. Yet to be reported by any group as the
Figure 1.5 Protein Nitrosylation Mechanism
during Apoptosis used as Example of
Physiological Relevance of Nitrosylation
during Common Cellular Events. Taken from
Mannick et al., 2002.
within the cell, and one might expect to
result of antioxidant treatment.
change in nitrosylation status with various
treatments. It would be logical to take this
experimental setup one step further and
attempt to detect methods of activating
and reversing protein nitrosylation,
especially in the context of current cancer
treatment and prevention regimens.
Because radiation causes apoptosis
through intentional/inadvertent
mitochondrial radical release, it would be
reasonable to expect a change in
nitrosylation levels as a result of
radiation. Conversely, antioxidants are
the cellular mechanism to quench radicals
a reduction in protein nitrosylation as a
7


1.3 Resveratrol
One of the earliest observations about the
cancer microenvironment was a state of hypoxia.
This discovery by Nobel Laureate Otto Heinrich
Warburg came to be described as the Warburg
hypothesis and has been the subject of much
research over the past 85 years. It was later
discovered that cancer cells actually prefer a
glycolytic metabolism, even in the presence of
oxygen suggesting that mitochondrial defects are
converting what little oxygen does get into the cell
into super-oxide and other damaging oxidant
species. Thus a suggested cancer treatment aimed at restoring the redox balance
within the cell is the addition of antioxidant therapy. Antioxidants taken as long-term
supplements have been
linked to prevention of
cancer, diabetes, and
obesity. (Houstis etal.,
2006) Among various
epidemiologic studies, one
of particular interest
regards the French
Paradox. This refers to
the phenomenon that the
French lead rather
unhealthy lifestyles (based
on tobacco and alcohol



HAQPH NW
Fig. 1.7 Mechanism of Oxidative Stress to Mitochondria and
Biochemical and Physiological Results. Taken from
Papaharalambus et al., 2007.
Resveratrol
Figure 1.6 Resveratrol Structure
and Illustration of One of the
Major, and Most Widely Known,
Sources of the Polyphenolic
Antioxidant. Taken from
www.resveratrolbenefit.org.
8


consumption, etc.) but Eire year after year on the lower end of nations in cancer
incidence per capita. Scientists have been trying for decades to understand the
potential causes of this paradox to prevent cancer as well as to search for novel
therapeutic agents. One of the areas of research has focused on the antioxidant
compounds found in red wine, a drink that the French consume quite liberally. Grape
skins are the source of many polyphenols that protect the grape and its seed from
environmental carcinogens. Antioxidant induction into tumor therapy regimens have
also been suggested to correlate with decreased progression/invasiveness and
reduction of metastatic potential. (Tang et al., 2008) Resveratrol, a natural product,
synthesized by a number of plant species, is an antioxidant that recently has garnered
attention for its potential involvement in the French Paradox. (Dudley et al., 2008)
Although a weak antioxidant itself, (Gusman et al., 2001) resveratrol has also been
correlated with mRNA and transcript
upregulation of several
intrinsic/mitochondrial antioxidants,
namely glutathione peroxidase (GPx),
thioredoxin reductase (TrxR),
selenophosphate synthetase (SPS), and
MnSOD. (Robb et al., 2008; Hu et al.,
2007, Baur et al., 2006) Resveratrol also
upregulates several genes related to cell
cycle regulation, differentiation, and
apoptosis, including p53, FAS, XIAP, and
BCL2, among many others. (Lagouge et
al., 2006; Aggarwal et al., 2004) A direct
target of resveratrol is the transcription factor and deacetylase, sirtuin 1 (SIRT1).
(Kaeberlein et al., 2006)
Exercise
or
Fasting Resveratrol
Figure 1.9 Resveratrols Known Direct
Interaction and Mechanism. Taken from Barn-
et al., 2006.
9


Resveratrol offers a potentially superior method of antioxidant administration,
as the drug not only acts as an antioxidant itself but also enhances the natural
antioxidant mechanisms of cells through transcriptional activation. (Gao et al., 2002)
Resveratrol has been shown to down-regulate growth factor receptor pathways in
vitro and in vivo. Of particular note, resveratrol has been shown to decrease iNOS
protein expression as well as intracellular NO release. (Roman et al., 2002) With
lowered levels of intracellular NO, the cell demonstrates less protein nitrosylation. It
remains unclear whether this decrease in nitrosylation is specific through targeted
mechanisms of resveratrol or due solely to its cellular and systemic antioxidant
effects.
1.4 Reasoning for Combining Radiation and Resveratrol
Radiation, although a potent killer of cancer cells, is limited in the extent of its
use due to the toxicity it poses on normal cells. Thus any adjuvant therapy that could
sensitize cancer cells to undergo apoptosis at a lower dose could spare damage to
surrounding tissues. It is logical to combine resveratrol therapy with radiation
treatment because of the commonality of action specific to the mitochondria. As
discussed above, radiation disregulates the mitochondria, in the process uncoupling
proteins of the electron transport chain as well as potentially altering proper function
of proteins in the p53 pathway. (Carew et al., 2002, Guzy et al., 2005) Resveratrol on
the other hand naturally restores mitochondrial regulation through its intrinsic and
downstream transcription factor induced antioxidant capacity.
Due to the large number of proteins and pathways that are affected by
resveratrol addition, (Aggarwal et al., 2004) it appears reasonable to credit a larger
biochemical effect beyond the single protein or pathway action typical of a majority
of biological research. This hints at an epigenetic change similar to DNA methylation
or acetylation but which acts on the protein level. Several groups have looked at
10


causal roles for combining resveratrol and radiation and each points to a different
protein product that is responsible for the effect of resveratrol/radiation synergy. This
lack of a common pathway or mechanism further adds to the argument of a systemic
epigenetic change. Scarlatti et al. (2007) show that resveratrol increases ceramide
concentration within prostate cancer cells, priming them for apoptosis by irradiation
while sparing surrounding cells and increasing the chances of retaining tissue and
organ function. Meanwhile Liao et al. (2005) propose that cell cycle arrest and NF-kB
are implicated in radiosensitization by resveratrol.
aki "**§* ept!
Transcription Factor*
morn to&fc yoMt ij
Cytokine*
fcbi CycfielU CrdkiAJ
C*cjf CjrifetU
Ceil cycle
TCP|
tort
Others
ifilii
Fig 1.10 Majority of Known Proteins Affected by Resveratrol Treatment Illustrating Vast Effects
of the Drug. This Suggests Epigenetic Change Associated With Treatment Rather Than Single
Targeted Effect. Taken from Aggarwal et al., 2004.
11


Non-lethal radiation doses illicit an effect similar to resveratrol. Many
proteins/pathways are affected that lead one to believe that there is an epigenetic
event caused by radiation. However when the radiation dose is increased to an
apoptosis-inducing lethal dose, this single outcome overshadows the other effects
observed at lower doses. The addition of resveratrol could mitigate the lower dose
effects while allowing for the DNA-damaging adducts caused by higher dose
radiation to serve their cell-killing purpose. Biochemically, this would be an ideal
method for controlling the side-effects of radiation, lowering toxicity and increasing
effectiveness of treatment.
1.5 Purpose of Thesis
The hypothesis of this thesis is that: radiotherapy can induce aberrant
nitrosylation events that lead to undesirable side effects and the addition of resveratrol
to radiation therapy may reverse these effects, leading to a more efficacious
treatment. There are three specific aims that this thesis hopes to demonstrate.
Specific aim 1: To show that resveratrol is synergistic with radiation treatment
by a number of quantitative assays that show strong synergy among a large majority
of tissue culture cell lines tested.
Specific aim 2: To demonstrate that the synergy between radiation and
resveratrol is at least in part attributed to changes in nitrosylation of proteins. This is
demonstrated by observing changes in nitrosylation of proteins via the biotin switch
assay coupled with Western blot analysis.
Specific aim 3: To establish that certain proteins are specifically targeted for
nitrosylation/denitrosylation by resveratrol and/or radiation by looking at gel bands
that show a strong change in expression upon Western blot analysis and analyzing
12


them further via mass spectrometry to elucidate their potential identities and
functions.
The purpose of this thesis is to demonstrate that protein nitrosylation is
induced by radiation therapy in the context of cancer treatment regimens, causing
(among others) inadvertent disregulation of: apoptotic pathways of cancer cells,
increased metastatic potential and/or primary tumor invasiveness. It will also be
shown that this deregulation can be reversed by using the antioxidant resveratrol as an
adjuvant therapy, resulting in a synergistic effect in apoptosis to cancerous cells in
vitro. This will be demonstrated using growth and inhibition assays to gauge the
synergistic reversal of growth in head and neck cancer cell lines plated and treated in
vitro. In addition, the biotin switch assay combined with gel electrophoresis will be
used to show a protein specific increase in nitrosylation with irradiation and a
decrease in nitrosylation with concurrent treatment of radiation and resveratrol.
Finally, specific proteins whose nitrosylation state changes with radiation and/or
resveratrol will be identified using mass spectroscopic analysis.
1.6 Scope of Thesis
The scope of this work is specific to treatments of cancer patients with
therapies in combination; as a result the work will examine the recently novel post-
translational modification of protein nitrosylation. Most treatments are simplistic in
their physiologic approach and have side-effects stemming from unwanted off-target
effects or cellular compensation mechanisms attempting to maintain homeostasis. As
is the case with radiation therapy, side effects are observed and novel therapeutics are
being tested to mitigate these off-target effects for a cleaner therapy.
As protein nitrosylation is believed to be an epigenetic change, the scope of
this work theoretically extends far beyond treatment of radiation treatment or even
cancer cell biology. Perfecting the biotin switch technique and discovering new
13


methods to accurately test protein nitrosylation in vivo can further the significance of
the nitrosylation change such that it is easier to observe and thus more readily
researched in the future.
1.6.1 Limitations of Data
As mentioned above, there are several pitfalls to the approach chosen for this
work. The use of in vitro assays with plated cells does not account for the tumor
microenvironment, which includes not only tumor cells, but the vasculature that
supplies oxygen and nutrients to tumor cells, and the supporting stroma. In addition,
the simplified two-dimensional plating mechanism is not indicative of a tumor
microenvironment and makes the dose of radiation delivered not physiologically
relevant.
The simplest method to test cellular mechanisms of therapy regimens is
through the use of cultured primary cancer cell lines in vitro. As such, several aspects
of tumorigenesis are altered in this type of model and are not completely indicative of
the in vivo tumorigenic system.
Interactions typical of tumorigenesis between cells of different types are not
detectable. Upon necrosis, cells release toxic signals to surrounding cells potentially
through mitochondrial burst that could be signaling through nitrosylation
mechanisms. This could a potential mechanism for in vivo resistance to radiation that
is not observed in an in vitro model. This would limit the proteins that are detected by
the biotin switch assay.
Another limitation inherent to the use of an in vitro model involves the
systemic pharmacology of drug absorption and modification. In vivo modifications
due to the digestion and filtration of resveratrol cannot be tested in an in vitro model.
14


Should this works promise be kept, mouse and other 3D models should be
investigated.
1.7 Arrangement of Thesis
This work will proceed in a style similar to that of a scientific journal article.
First the background of cancer biology, protein signaling mechanisms, resveratrol and
radiation treatments will be discussed, the hypothesis, purpose and scope described
above will tie the background together and give an idea as to why one chose to
research this topic and why these two treatments were added in combination. Next the
paper proceeds to outline the materials and methods used to test the hypothesis, why
these particular methods were chosen and what are the pitfalls and successes of these
experimental methods in general and specifically in this work. Data are provided to
the experiments performed, from which a discussion is extrapolated and conclusions
are drawn.
15


2. Materials/Methods
2.1 Materials
Tissue culture cell lines used for this project were all of various head and neck
(H&N) phenotypes. UMSCC2, UMSCC8, UMSCC10B and UMSCC22A cell lines
were a gift from Dr. Thomas Carey of the University of Michigan. MDA1483,
MDA1586, and MDA584 cell lines were a gift from Dr. Scott Weed of West Virginia
University. HN19 cell lines were obtained from MD Anderson via Dr. Antonio
Jimenez at the University of Colorado. FaDu, Detroit562, SCC25 and CCL30 cell
lines were purchased from American Type Culture Collection (ATCC). Fetal bovine
serum, Dulbeccos modified eagle medium and trypsin were purchased from
Mediatech at their on-site supply center at the University of Colorado-Denver
Anschutz Campus. Petri dishes and flasks were purchased from LightLabs. Sodium
dodecyl sulfate (SDS), sodium ascorbate (NaAsc), HEPES, neocuproine, triton X100,
resveratrol, ammonium persulfate (APS), dithiothreitol (DTT), iodoacetamide, methyl
methanethiosulfonate (MMTS), and L-glutathione were purchased from Sigma
Aldrich. Glycine, Tris base, Ethylenedeamine tetraacetic acid (EDTA), EZ-link
biotin, NeutrAvidin agarose, methanol, and acetic acid were purchased from Fisher
Scientific. Mineral oil, glycerol, protein assay kit, silver stain plus kit, acrylamide,
readystrip IPG strips were purchased from Biorad. The irradiator used was an
RS2000 model (Rad Source Technologies). The isoelectric focusing equipment used
was the IEF focusing cell model produced by Biorad. Calcusyn software made by
BioSoft was used to calculate combination index values. The mass spectrometer that
was used for protein identification was a 6300 LC/MS Ion Trap with nanospray
16


source from Agilent. The liquid chromatography (LC) setup was equipped with
Agilent nanoLC and capLC pumps and an autosampler.
2.2 Cell Culture
All cell lines were grown in DMEM culture media with 5% heat inactivated
fetal bovine serum (FBS-HI). Antibiotics were not used as they can cause undesirable
drug/drug interactions or other unknown side-effects. Cells were split twice weekly
either 1:5, 1:10 or 1:20 depending on confluency using 0.25% Trypsin/EDTA in
DMEM.
Cells were maintained in sealed filter-cap 75cc flasks. All flasks were kept in
a single digitally regulated incubator, temperature controlled at 37C, humidity at
95% and with a constant CO2 concentration of 5%.
2.3 Resveratrol/Radiation Treatment
2.3.1 Generic Procedure
In all experimental treatments, resveratrol was added the day before cells were
harvested. Resveratrol was added at the peak of its synergistic concentration range
which corresponded to physiologic levels observed in mouse and other in vivo
pharmacokinetic models. In in vitro culture models this was 10pM final
concentration. The media that the cells were grown in was removed and media with
fresh growth factors and resveratrol at the prescribed concentration was added in its
place. The following day, cells were irradiated 1-2 hours before being harvested.
Radiation was also performed at a concentration that corresponded to peak observed
synergy with resveratrol determined during this work (4Gy). Although this
concentration cannot be compared to physiologic amounts because the monolayer
culture is not comparable to what patients or rodents receive in in vivo models or
actual treatment scenarios, this corresponded to the dose that actually reaches the
17


target tissue in 3D models using approximations for decay corresponding to decay
rate through animal tissues.
2.3.2 Specific Protocol
In order to develop the most synergy as well as to maintain consistency of the
time course of treatment, experiments were performed to determine the best time
course to be taken such that optimal results would be observed. Per standard
operating protocol, it was determined that drug treatment should occur roughly 24
hours before radiation such that early and delayed proteins would have a chance to be
expressed or altered before treatment with radiation. Signaling from radiation
treatment is of the early type, such that only an hour or two are required for a change
in protein expression. This seems appropriate from the point of view that cells hit
with radiation cannot proceed with cell replication unless they are immediately
repaired. Should more than an hour or two been taken after radiation treatment, cell
death would have been observed which corresponds to protein degradation and a
decrease in yield from the quantity of plated cells used.
The irradiator was previously calibrated, however the special dose received
did vary as the irradiator was not a linear accelerator. A lead brick was used to
channel the ionizing rays through the small space in which the cell culture dishes
were located.
2.4 MTT/Inhibition Assay
2.4.1 Generic Procedure
To determine synergistic effects of resveratrol and radiation on inhibition of
cell viability, tissue culture cells were plated in 96 well plates at 4000 cells/well
(lOOpL) after being broken apart into a single cell suspension by repeated aspiration
with a 20 gauge needle. The following day cells were treated with resveratrol in final
18


concentrations that varied from .03mM to 100pM and irradiated the next day with a
radiation dose from 2Gy to 6Gy. Plates were taken down 120 hours after initial
resveratrol addition by incubating with tetrazolium salt at 2mg/mL for 4 hours,
followed by resolubilizing the solution with 73% alcohol, 2% HC1 and 25% H2O and
reading the 96-well plates on a plate reader at 450nm. Synergy was determined by
calculating Combination Index (Cl) values using the BioSoft software CalcuSyn
described in detail below.
2.5 Ckmogenic/Proliferation Assay
2.5.1 Generic Procedure
As an alternative to cell viability assays, clonogenic assays were also
performed. Singly suspended cells were plated at lOOOcells/well (5mL) in 6-well
plates, resveratrol was added at final concentrations of 6mM, lOmM and 30mM on
the following day and the plates were irradiated at 2Gy, 4Gy and 6Gy the next day.
Cells were allowed to grow for 10-14 days after resveratrol treatment until the
resulting colonies could still be distinguished. Plates were taken down by fixing cells
with reagent grade methanol and staining cells with crystal violet stain. Colonies were
both counted and combination index values calculated to demonstrate synergy as well
as photographed for visual representation.
Calcusyn (Biosoft) uses Chous median-effect equation/principle to determine
significance of dosing and effect. Chous dose-effect relationship can be written:
where D is the dose, Dm is the median-effecting dose, fa is the fraction affected by the
dose, fu is the fraction unaffected by the dose and m is the exponent signifying the
Eq. 2.1
19


sigmoidicity of the dose-effect curve. From this equation, the confidence interval (Cl)
can be defined as:
a=^ + l£k + J£M£^ Eq2.
(D;c)l (Dx)2 (Dx)l(Dx)2
In which Di and D2 are two mutually non-exclusive drugs that have totally
independent modes of action. The first term in equation 2.2 accounts for the effect of
the first drug alone, the second term accounts for the effect of the second drug alone
and the third term accounts for the drugs actions in combination. Thus when
compared to plots of individual drug dose-effect relationships, the combination index
can account for dose response in addition to individual effects. A combination index
value greater than 1 indicates an antagonistic effect, equal to 1 indicates an additive
effect and less than 1 indicates a synergistic effect. The value is considered
moderately synergistic if it is between 0.7 and 0.85, synergistic if it is between 0.3
and 0.7, strongly synergistic between 0.1 and 0.3 and very strongly synergistic <0.1.
In order for a combination index to be derived, each drug alone should have a dose-
effect relationship. At least three or more data points for each single drug were used
as required by Chous method and each of these three concentrations were combined
with three concentrations of the other drug.
2.6 Protein Assay
2.6.1 Generic Procedure
Protein concentrations were determined using the Biorad protein assay kit.
5pL of protein sample/standard were used per well in a standard 96 well plate. lmL
of reagent A was mixed with 20pL of reagent S, 25pL of which was added to each
well. 200pL of reagent B was added to each well and plates were read after 15
minutes at room temperature. A standard curve was used ranging from .18 to
1.44mg/ml of protein in 2x dilutions increments. Protein samples were diluted either
20


5 or 20 times depending on initial sample concentration so that they would fall within
the range of the standard curve. All standard and unknown samples were performed
in triplicate.
2.7 Biotin Switch Nitrosylation
2.7.1 Generic Procedure
The biotin switch assay was performed as originally described by Jaffrey et al.
(2001) with several modifications. Cells were initially plated in 1500mm petri dishes
until they reached at least 90% confluency. After confluency was reached, cells were
treated with 10pM
2.0
resveratrol, and/or
followed 24 hours later
by 4 Gray of radiation,
four treatment groups
were created: a control
group (no drug or
radiation), drug alone,
radiation alone, and-
combined radiation and
drug treatment in the
timecourse described.
Taken from Hirsch et al., 2002. Cells were lysed 1-2
hours following radiation using scraping and 500pL HEN Buffer (25mM HEPES,
ImM EDTA and O.lmM neocurpoine) per 150mm dish and cells were sonicated for 3
rounds of 3 pulses each to liberate membrane bound proteins. A protein assay was
performed using Biorads RC DC protein assay kit and volumes were adjusted to
make protein levels constant at lOmmol/mL. Proteins were stored at -80C if the
Wavelength (nml
Figure 2.1 Spectroscopic Reading After Reaction of L-Cysteine
with Sodium Nitrate Demonstrating Creation of Nitrosylated
Cystein. Used to Confirm Desired Product and Determine Product
Yield. Maximum Absorbance Reading from CysNO at 334nm.
21


protocol did not continue past this point. Proteins were incubated with 40pM NO
donating agent (GSNO or L-CysNO) or control (GSH or L-Cysteine) for 60 minutes.
Free cysteines not on the surface were blocked by denaturing the proteins. This was
done by heating the proteins in a heat block at 50C in a solution containing 2.5%
SDS and 20pM MMTS. This also had the effect of creating disulfide bonds out of the
free internal cysteines. Following the denaturing step, excess donating agent and
MMTS were removed by spinning down using Microcon columns from Millipore
with a nominal molecular weight limit (NMWL) of 3,000 Daltons at 14000g for 100
minutes at 4C. After the remaining proteins are brought up to lOOpL with HEN
buffer containing 1% SDS, 50mM NaAsc and lOmM Biotin-HPDP, proteins were
incubated in the dark at room temperature with intermittent vortexing. The excess
biotin was removed by spinning down using new microcon desalting columns at
14000 x g for lOOmin. Volumes were adjusted to lOOpL with HEN buffer. 2 volumes
of neutralization buffer (30mM HEPES, pH 7.7,100mM NaCl, ImM EDTA, 0.5%
Triton X-100) were added as well as 15pL neutravidin-agarose/mg protein used in
initial protein sample. Samples were then incubated with the neutravidin-agarose
resin for one hour at room temp. This was followed by washing at least 4 times with
10 volumes 600mM NaCl neutralization buffer with vortex pulses between washes
with brief spin downs. Bound proteins were eluted in 50pL neutralization buffer
containing lOOmM 2-merceptoethanol and without Triton X-100. Final solution
preparation depended on which western blot analysis was being used. For ID gels,
IOjiL of 5x Laemmli loading buffer (0.25M Tris pH6.8,40% glycerol, 4g SDS, 14mg
bromophenol blue [BPB]) was added to each sample and heated to 95C for 10-15
minutes to elute the proteins from the beads in the resin. For 2D gels, the volume
brought up to 300pL with active rehydration buffer solution described in the
isoelectric focusing section below.
22


2.8 Isoelectric Focusing
2.8.1 Generic Procedure
Isoelectric focusing was performed on Biorads IEF focusing cell. Proteins
were loaded onto 17cm IPG strips using the active rehydration protocol provided with
the strips. 300pL of protein in active rehydration buffer (8M Urea, 4% CHAPS,
lOOmM DTT, 0.2% w/v Biolytes, 0.001% BPB) was added to the focusing tray,
followed by the EPG strips and covered with mineral oil in each well to prevent
evaporation. The rehydration took place by conditioning the strips at 50V for 12
hours. Isoelectric focusing proceeded with a 250V conditioning step for 15 minutes
following by ramping the voltage up to a maximum of 8,000V with the current not
exceeding 50pA/strip. This maximum voltage is lower than that suggested 10,000V
because it helped ease the arcing that was observed at the higher voltage. Final
voltage passed through the strips was 60,000 V hrs. Finally the strips were held at
500V to prevent diffusion of focused proteins. Following focusing, gels were
equilibrated for 10 minutes in each equilibration buffer to minimize streaking and
other artifacts upon running of the gels. Equilibration buffers I and II were prepared
by mixing 6M Urea, Q.375M Tris-HCl pH 8.8,2% SDS and 20% glycerol. Buffer I
required the addition of 2% w/v DTT and buffer II required addition of 2.5% w.v.
iodoacetamide. 6mL was made up of each buffer and split equally between each well.
2.9 SDS-Polyacrylamide Gel-Electrophoresis (PAGE)
2.9.1 Generic Procedure
The protocol for running ID or 2D gels varied very little. Polyacrylamide gels
were either made or purchased depending on what size/gradient was needed. Most of
the gels were hand-made to save on cost.
23


Glassware was scrupulously washed to ensure that no protein residues
remained on the plates for the purpose of having clean gel bands during mass
spectrometry analysis. Scrupulous washing entailed cleaning two times with soap, the
first with deionized water and the second time with deionized distilled (MilliQ) water
at a purity of 12-180 resistance. All glassware was dried with kim-wipes to minimize
drying time and potential for contamination of anything in the air that might settle on
a wet surface. After completion of washing and drying, the glass plate openings were
covered with kim-wipes until use to further protect from proteins in the air as dust
such as keratin which often contaminate gel-extracted bands.
Using the Biorad gradient forming kit, between 4 and 8 gels were
simultaneously made by combining almost equal amounts of light and heavy gradient
acrylamide gel mixtures. The heavy gradient mixture contained a slightly larger
volume to account for tubing and still allow for the bottom of the gel to be made with
the desired concentration of acrylamide. The light gradient mixture contained a final
concentration of 8% acrylamide, 0.375M Tris pH 8.8, and the remainder of the
volume was made up of deionized distilled H2O. In addition polymerizing and
denaturing agents were added in the following final volumes: 0.05% APS, 0.025%
TEMED and 0.1% SDS. The heavy gradient mixture was made using a final
concentration of 16.5% acrylamide, 0.375M Tris pH 8.8, 10% glycerol (for weight),
and the remaining volume was made up of deionized distilled H2O. In addition
polymerizing and denaturing agents were added in the following final volumes:
0.05% APS, 0.025% TEMED and 0.1% SDS. The light and heavy gradient mixtures
were added to separate containers and after opening the valve to fill the gel casting
chamber, the gradient mixtures were allowed to slowly mix. Thus while the light
gradient mixture was slowly pouring into the casting chamber, the heavy gradient
mixture was introduced to create an acrylamide gradient. This non-linear gradient gel
forming method made it possible to minimize the number of gels being run by
24


making it possible to run both large and small proteins could be run on a single gel.
After the gels reached the desired height, they were allowed to polymerize for 20-30
minutes. During this time, gels were covered with H2O saturated butanol so that the
top layer of the gel would remain smooth. The H2O saturated butanol was removed
before proceeding. Depending on whether a ID or 2D gel was used, the gel-casting
procedure varied from that point forward.
For ID gels, an upper stacking polyacrylamide gel was poured around an 8-
well comb. The stacking gel was made with the following final concentrations: 4%
acrylamide, 0.375M Tris pH 6.8, remaining volume of H2O, 0.1%APS, 0.05%
TEMED and 0.2% SDS. Samples were loaded along with an unstained standard
ladder into individual wells and were run at a constant 200V for 50 minutes.
For 2D gels, 1% low-melt agarose mixture with 0.05% BPB was loaded over
the gels. IEF strips were then loaded over the completed gels/into the agarose
stacking gel as flush as possible to the running gel. 2D gels were run at a constant
current of 0.05Amps per gel for 12 hours or until the BPB from the agarose stacking
gel came close to the bottom of the running gel.
2.10 Silver Staining
2.10.1 Generic Procedure
Silver staining followed the Biorad silver stain plus kit protocol essentially as
described by the manufacturer. After gel electrophoresis, proteins were fixed to the
gels overnight. Although only required to affix for 20 minutes, better signal to noise
ratio was observed upon significantly increased fixation times. 400mL of fixative
enhancer solution was used per large gel and 200mL per mini gel. Fixative enhancer
solution consisted of 50% reagent grade methanol, 10% reagent grade acetic acid,
10% fixative enhancer concentrate and 30% deionized distilled water. Each gel was
25


rinsed 2 times for 5 minutes each (no longer than 10 minutes) with 200mL of
deionized distilled water regardless of size with constant pouring of water on top of
the gels and gentle shaking so that consistent washing could be achieved across the
entire surface of the gel. Staining was performed by adding lOOmL of staining
solution to each large gel and 50mL to each mini gel. Prepared right before use,
lOOmL of staining solution consisted of 35mL deionized (but not distilled) water,
5mL silver complex solution, 5mL reduction moderator solution, 5mL image
developer reagent, and 50mL developer accelerator solution mixed in that order. The
developer accelerator solution should be heated to room temperature before use. Gels
were developed for 10-14 minutes, or until the protein standard reached desired
darkness. The reaction was stopped by adding 5% acetic acid to the reaction vessel
for 15-20 minutes. Gels are then stored in deionized water at 4C until bands/spots
are excised for mass spec analysis.
2.11 Mass Spectrometry
Mass spectrometric analysis of in gel digested proteins was performed by the
mass spec core facility at the University of Colorado Health Science Center. Gel
bands/spots were excised using cut lmL pipette tips and stored in ependorff tubes
containing 500pL distilled H2O in -20C until mass spectrometric analysis was
performed.
Upon removal of samples from the -20C freezer, samples were digested in
trypsin to remove the proteins from the gel as well as to breakdown proteins into
smaller pieces, more readily read by the mass spectrometer. The samples were also
treated with sodium thiosulfate and potassium ferricyanide solutions in order to
remove the silver adducts associated with silver staining of gels. An LC/MS Ion Trap
mass spectrometer was used for this experiment. All peptide sequences identified,
potential protein names, and their associated confidence intervals are presented.
26


3. Results/Discussion
3.1 Clonogenic/Inhibition Assays
As an initial step to assess the potential of resveratrol to synergize with radiation, in
vitro assays with a panel of head and neck squamous cell carcinoma cell lines were
done. Both inhibition and growth assays were used because of limitations with each
experimental setup. Each setup tests a different parameter of cellular growth, taken
together and with other in vitro growth assays show a more complete picture of how
resveratrol, radiation and combined resveratrol/radiation treatment affects cancer cell
growth.
The MTT assay measures the mitochondrial activity of cells following
treatment by reduction of tetrazolium salt to an insoluble formazan compound. When
a cell is signaled for apoptosis or necrosis, cellular metabolism stops and thus
mitochondrial activity drags to a halt. Thus this assay directly measures a cells
metichondrial capacity to metabolize tetrazolium salt at a certain time point after
treatment. Cells are plated at high concentrations on the wells such that they reach
confluency in control wells at the end of the assay. This is because the assay
disregards cells abilities to stop replication, repair DNA and then continue replication
to form colonies, only the amount of cellular metabolism is accounted for. As shown
in figure 3.1, Cl values for all physiologically relevant doses were well below the 1.0
mark, demonstrating strong synergy in all cell lines.
27


A.
1:3 1:5 1:15
X 1:30 + 1:50 1:1.5
Fig 3.1 Radiation and resveratrol are synergistic when assess by MTT assays. A. Isobologram for
FaDu cell line showing combination indices for combination of radiation and resveratrol. Points below
line represent synergistic effects. And B. bar chart showing combination index (Cl) values for all cell
lines at various resveratrol concentrations in combination with 4 Gy radiation. All Cl Values are below
1.0 additive mark, suggesting systematic synergy.
28


Synergy values are reported for all head and neck cell culture lines throughout
the work. Combination index values less than 1 indicate synergy, equal to 1 indicate
additivity and greater than 1 indicate antagonism. Results of the MTT assay show
moderate to strong synergy values across all cell lines reported. The 1483 cell lines
was chosen to continue on to the clonogenic assay because it showed the strongest
synergy by MTT and the phenotype of 1483 cells is conducive to simple colony
formation when plated on a monolayer. The results for the clonogenic assay were
antagonistic, contrary to what was expected of this assay. A discussion follows as to
reasoning for unexpected experiment outcome for clonogenic assay.
Figure 3.2 shows the results of clonogenic assays using cell line 1483 and
treating with radiation in combination with resveratrol. Treatment was performed as
described above at concentrations listed above each column and row. Colonies were
counted and half of each well was spotted with felt pen for ease of observation if
colonies exceed 50 cells. The number of colonies was counted and Calcusyn (Biosoft)
was used to calculate synergy. This data is presented above. The subsequent page
shows examples of nondescript colonies that complicated interpretation of the
clonogenic assay.
29


A.
3:1 1.5:1 1:1
X 5:1 + 2.5:1 1.66667:1
Resveretrol Cone (|iM)
Fig 3.2 Resveratrol and radiation are additive or antagonistic when measured by clonogenic assays in
the 1483 cell line. A. Isobologram of clonogenic assay showing additivity or antagonism at various
concentrations. B. CIs of resveratrol at 6, 10, or 30jiM in combination with 4 Gy radiation. The Cl
values are higher than 1.0, indicating a mild antagonistic effect. Systemic sources of error within
clonogenic assay are likely at fault.
30


The clonogenic assay is considered the gold standard of clinical radiation
oncologists to determine cell survival. It is a more direct measure of a cells ability to
replicate, however the methodology is still flawed. Individual cells are plated at very
low concentrations and colonies of 50 cells or more are measured at the end of the
assay. Because of the low concentrations at which cells are plated as well as the
plating method which ensures a single cell suspension, cells spread far apart within
the wells and must replicate at least six times in order to be counted as a colony.
For a cell to replicate six times after it has been treated with radiation and/or
resveratrol it must have repaired the DNA damage that was caused by treatments, as
well as resumed its cell cycle. Although this is a more representative method of
looking at cellular response to treatment, there are still several flaws in this assay that
resulted in a Cl value that was higher than expected and contradicted what others
have observed for the same assay performed on the same cell line. This demonstrates ,
the necessity to properly setup an experiment as well as the necessity to be able to
know how experiments are setup in primary literature instead of just looking at a data
value.
Further observations of clonogenic plates indicated several-factors that might
have confounded the data. Figure 3.4 demonstrates the difficulty in scoring colonies.
An important variable to control with the clonogenic plates was how long the cells
were allowed to grow. Usual doubling time of the 1483 cell line is approximately 24
hours, which is relatively fast compared to other cultured lines; but treated cells
require time to stop the cell cycle, repair DNA damage and restart the cell cycle.
Furthermore it is not known whether the doubling time is affected by treatment, a
detail that should be investigated further. Assuming that this is ignored, there are still
several sources of error within the clonogenic assay that should be addressed.
31


The length of time that the assay is left to run and the size of the wells are the
most important variables of the experiment. In control wells and wells with low doses
there is a large chance that the colonies will grow together and will erroneously lower
the colony count in the control wells. This phenomenon was observed on several
occasions due to the size of some of the colonies observed. For a single cell to grow
to such a large colony would require much longer than the two weeks maximum
allowed for these experiments to run. In addition, some colonies were observed that
although were not excessively large, showed cells that had a different appearance
under the microscope (either based on clear differences in cell size, roundness, etc.).
This observation suggests that two separate colonies grew together. Jointly, these
observations suggest that either the number of cells plated per well size used needs to
be reduced for each treatment group or larger wells need to be used with the same
initial number of cells per well used here.
Another observation regarding accuracy of the clonogenic assay invol ved a
different parameter with regard to colony size. By the end of the experiment, the
wells that were treated with high doses of one or both treatments had very few
colonies. But more importantly the colonies did not look like the colonies of the
lower dose treatment groups as demonstrated in Figure 3.3 parts c through f. At
higher doses, the cells of the colonies were not adhering tightly to one another. The
cells also appeared to take on a different phenotype. This can potentially be explained
by the phenomenon of genetic instability commonly observed in cancerous cells.
When DNA damage is incurred by cancer cells, the cell undergoes cell cycle arrest in
order to repair the damaged DNA before it continues to replicate. This halt to cell
cycle progression can be erroneously overcome if the process of cell cycle arrest fails
to occur, or occasionally conflicting cell signals override cell cycle arrest and signal
for proliferation to continue. However, with varying amounts of adducted DNA, the
cell cannot successfully separate its DNA during anaphase and the result is a large
32


entanglement of cytoplasm that appears to be a multinucleated cell (or a cell that has
gone through several rounds of DNA replications but that cannot completely separate
its cytoplasm because the DNA has not separated). This would at least partially
explain the loss of adhesion and the observed phenotype of cells at higher treatment
doses.
33


Figure 3.3 Colony Formation In Clonogenic Assay Due to Individual and Combined Treatment Regimens. Wells
were observed under high powered microscope and the left half of the well was spotted with a marker to better help
visualize coloniesVisual Demonstration of Inhibition of. The right side of each well was left unmarked for
comparison.


Fig. 3.4 Snapshots of cells after various combination treatments in clonogenic assay. A. Colony
clearly too large to have derived from single progenitor cell in just 10 days. B. Colonies that
havent yet fused, but with different refractory levels allowing them to be separated had they
fused. C. Fused colonies still differentiable. D. High radiation dose results in some migratory
phenotype observed. E. More motile phenotype observed as a result of higher dose of radiation. F.
Still higher rate of mesenchymal phenotype observe, here clearly demonstrating connections
between cells as a result f DNA bridges that have failed to break and allow for complete
cytoplasmic separation.
35


(A) (B)
Fig 3.5 Comparison of normal and damaged DNA. Bright spots indicate telomeres at the ends of
each chromosome clearly separated in A, whereas in B the DNA is in a large disordered bundle
without orderly telomeres capping each chromosome.
Another possible explanation for the observed change in morphology of the
cancer cells at higher treatment doses is a loss of proteins that allow for cell binding
to each another. This can happen for a number of reasons, the most intriguing of
which is that radiation signals cells to undergo epithelial to mesenchymal transition
(EMT). This phenomenon has been reported by many groups in numerous models
(Jung et al., 2007) and is an alarming effect of radiation therapy. If during treatment
the cells gain increased metastatic potential, this would definitely be an unwanted
side effect of radiation therapy. This study was not designed to address the addition of
resveratrol in reversing the effect of radiation-induced metastasis. Cells that reverted
to an epithelial phenotype would have undergone apoptosis and would not have been
seen. Furthermore, it is very difficult to quantify this phenotypic change especially in
a small number of cells. Thus in the context of this experiment, it is difficult to
accurately enumerate on EMT beyond a brief explanation.
36


Radiation-induced EMT is not something measured by clonogenic assays, but
is definitely deserving of future study. This does however present an interesting
ancillary note, as a potential method by which vertebrates could use nitrosylation
signaling to their benefit. It is known that DNA methylation identifies which copy of
the DNA is the original and which is the newly synthesized strand. This allows for the
cell to distinguish so called stem cells from their progeny. As such, there is evidence
to suggest how selective pressures incorporated this sort of conservation mechanism
against mutations. In a similar manner, protein nitrosylation could serve as a facile
method for cells to correlate cellular metabolic rate with motility, or wound repair
with motility. Mitochondria release low levels of nitric oxide at high metabolic rates.
This happens when the organism is growing, a condition in which increased motility
of cells is beneficial to an increased growth rate. In addition, when an injury occurs or
when the organism has undergone intense exercise, the damaged (In either case)
tissue also experiences increased metabolic rate in order to repair the damaged tissue
and would benefit from a change in protein expression/action due to nitrosylation. In
exercise physiology, researchers have actually seen correlation between nitric oxide
production and a phenomenon known as delayed onset muscle soreness (DOMS).
DOMS is the muscle pain felt two to three days after exercise that seems to be
unrelated to fatigue, but has been found to correlate with the extent of muscle damage
after high intensity exercise. Both of these examples demonstrate how this epigenetic
change, like other aberrant changes in cancer, is a typical action of normal cells taken
out of context of normal tissue function.
37


3.2 Biotin Switch/Gel Electrophoresis/Silver Staining
The majority of the time spent researching this project involved optimizing
the biotin switch and gel electrophoresis techniques to work with each other. From
the onset of the project, it was clear that this was going to be the most challenging
piece of this project. Not only is the biotin switch method relatively new, having been
originally published by Jaffrey et al. in 2001, but it has been criticized extensively in
review articles and many groups have published enhancing or counterclaiming
protocols, and even anecdotes about lab groups switching buildings only to observe
that the biotin switch protocol failed to work properly in their new laboratory.
By its very nature, protein nitrosylation is a difficult phenomenon to measure.
This post-translational modification is a covalent addition that depends on the
oxidation state of the proteins in question and is thus reversible. The key to
assessment of nitrosylation events is to either measure the phenomenon in real time or
replace the reversible reaction with a non-reversible one that is both representative
and easily measureable. The biotin switch is an example of the latter course replacing
a sulphur-nitrogen bond with a disulfide bond, on one end of which is a biotin
molecule that can be bound to streptavidin beads, allowing for the separation of
nitrosylatable proteins from non-nitrosylatable proteins.
The method by which this reaction proceeds is at the root of problems with the
methodology. The initial difficulty encountered when starting this protocol was the
choice of nitrosyl donating agents. Previously literature suggested three different NO
donators, nitrosylated glutathione (GSNO), nitrosylated Cysteine (CysNO) and S-
nitrous-acetylpenecillamine (SNAP). Jaffrey et al. (2001) originally chose GSNO
because it is widely recognized as the significant in vivo NO donor and thus
represents the most physiologically relevant donating agent. This group also used
SNAP as an alternative but showed that nitrosylation was not as significant with this
donor molecule. Later research however compared CysNO and GSNO in order to
speed up the biotin switch assay. It was found that the kinetic rate constant of CysNO
38


interacting roughly five times that of CysNO (Goldstein et al. 1996). Researchers
used this literature to support their findings that interaction of CysNO instead of
GSNO improved their assays functionality without sacrificing its specificity
(Forrester et al. 2007).
The next challenge faced by researchers trying to implement Jafffeys Biotin
Switch was optimizing the oxidation reduction steps of the protocol. Several groups
observed concentration dependence of various reagents beyond those observed by
Jaffrey as well as specific details omitted from Jaffreys protocol altogether. Several
groups commented on the specificity of ascorbate in reducing only nitrosylated
cysteine residues while supposedly not having the reducing power to alter disulphide
bonds or other post translational modifications. If these modifications are altered, it
would leave an exposed modifiable non-cysteine amino acid residue that could
provide false positive results if then reacted with the nitrosylated biotin marker.
(Forrester et al., 2007, Giustarini et al., 2008, Huang et al:, 2005, Zhang et al., 2005)
The redox steps were further complicated because of the dependence of these
reactions on ions in DI water that were omitted from the protocol. In particular, Wang
et al. demonstrated that not only was copper necessary during the ascorbate reduction
step as ascorbate does not itself reduce sulphurs but requires the presence of metal
cations. This was further enhanced by the use of EDTA in the solution. This metal
chelator is often used because of the undesirable effects of metal ions in solution but
in this case it was necessary to not use EDTA (or other similar chelating agents such
as DTP A) for they would not allow for the reduction of disulfide bonds by ascorbate
and subsequently would not allow biotin binding to the formerly nitrosylated cysteine
residues. Wang claimed that Jaffrey was inadvertently contaminating glassware or
other lab supplies with copper and thus achieving results, but that better sensitivity
while maintaining specificity could be obtained if copper was added to the
ascorbate/biotin solution directly.
39


Still others have claimed that biotin is not the best conjugate to use in order to
connect streptavidin beads to nitrosylated proteins because of its irreversible nature
and in conducting this work a similar observation was noticed. Biotin affinity for
streptavidin is one of the highest known between two organic molecules. However
this becomes a problem when one attempts to separate proteins from bound
biotin/streptavidin in order to run down a gel via electrophoresis. Hirsch et al.
discovered novel biotin-like molecules that have lower affinity for streptavidin. This
doesnt sacrifice the strong binding power of biotin for streptavidin but does allow for
their separation i 17when needed and thus improves yield.
It was mentioned above that there are two methods for measuring
nitrosylation levels, 1) the replacement of nitrosyl group with a more stable
irreversible bond such as a disulfide bond, of which the biotin switch is an example
and 2) recently published work by (Santhanam et al., 2008) focuses on an alternate
method of nitrosyl group identification and in particular quantification. The selective
fluorescent labeling of S-nitrosothiols (S-FLOS) method of detection is similar to the
Biotin Switch in its first few steps of free cysteine blocking and nitrosyl group
removal by ascorbate, but instead of labeling the reduced sites with a biotin marker to
separate out the proteins that are later connected to streptavidin beads, the S-FLOS
method simply replaces the S-NO group with a fluorescent Cypro-Maleimide dye.
Although this doesnt allow for protein separation by attachment to beads, this
protocol permits proteins be separated by technologies such as flow cytometry or
simply run on a gel in the presence of non-nitrosylatable proteins; allows for the
localization of these proteins within the cell as a method by which to verify correct
protein identification and allow for quantification of nitrosylation with a higher
degree of sensitivity. This technology was not used in this work for a couple of
reasons. Not only was it more expensive to use fluorescent labels, but mainly because
it would be impossible to prove the nitrosylation of removed gel spots/bands if
40


background proteins were not removed prior to running of proteins on gels.
(Santhanam et al. 2008)
As mentioned in the materials/methods section, running two-dimensional gels
requires first separating proteins based on charge by isoelectric focusing, followed by
separation on a second dimension according to size. Protein isoelectric focusing
requires the application of a very large voltage to drive proteins through an
immobilized pH gradient
to the proteins
Biotin Switch
Btotin-HPDP >*ij
LABEL
REDUCED
SITES
*
MMTS
? *
?
-
coo*
BLOCK
FREE SITES
REOUCE
SNO SITES
1 z**-**1^*
8*TJ - BUD TO
, AVI DIN
f
s
i
LABEL
REDUCED
SITES
---------
Cv-MtKlmld*
OTT Elute
kj 2D Cel
S-FLOS
Fig. 3.6 Comparison of Biotin Switch and S-FLOS Nitrosylation
Labeling Schemes. Taken from Santhanam et al., 2008.
isoelectric point.
Achieving this large
voltage, often up to
10,000V was difficult to
accomplish and
troubleshoot. Several
potential causes were
examined that could
have affected IEF
voltage. The primary
suspected problem to successful isoelectric focusing was increased salt concentrations
as a result of IEF strip preparation that promoted increased temperature and
subsequent IEF strip overheating and burning the strips. This burning of the strips
makes it impossible for proteins to separate through them, as well as posing amild fire
hazard for the lab. Preparing the proteins before adding them to the IEF strips
involved washing with high concentrations of sodium chloride solution that was
difficult to remove, even upon washing the IEF strips in running buffer solution that
contained minimal salt. Optimizing the last washing steps of the biotin switch
protocol to properly function with the low salt requirements of the isoelectric
focusing machinery proved to be the crux of the protocol, one that was never
41


consistently overcome and which forced the researchers to settle for simple one-
dimensional gel electrophoresis, an analysis which provided much lower separation
of potential protein products.
The first and simplest problem encountered when performing these
experiments was a failure to observe any bands upon silver staining. It was here that
the researcher first discovered that water MilliQ water isnt always preferable to DI
water or tap water. The protocol for silver staining calls for silver solution to be added
to gels which is then allowed to slowly be reduced, forming elemental silver
conjugates onto protein bands within the gel. This reduction step is facilitated by
metal ions within the water, which if absent cannot provide their very important
function. The lack of metal ions in MilliQ water resulted in no protein detection.
Another lesson learned by performing silver staining can be summed up with
the classic saying keep it simple. The original staining protocol that was used was
developed in house and required many steps, often with exact timing. This proved
difficult to achieve when staining up to eight gels at the same time and very long
when these gels were run sequentially. The final protocol chosen for staining was the
simple BioRad protocol originally developed by Merril et al. in 1981 that allows for
staining in three steps, takes only a couple of hours and is not sensitive to changes in
timed steps within the protocol. This protocol worked very well, allowing for even
faster times than the written protocol suggested without sacrificing quality of results.

42


o
250kD #*
150kD t-
lOOkD
7SkD^|
50kD
37kD
f 4
Bands excised for mass
Spectroscopy analysis
Figure 3.7 Silver-Stained SDS-PAGE demonstrating protein nitrosylation in treated and
untreated cells. Differential patterns excised for mass spec analysis labeled numerically.
Although eleven bands were excised for mass spectrometry analysis, only four
were tested at this time. These bands corresponded to the proteins of most interest,
those that had the most significant changes in expression across various treatments
and were thus the most relevant to this work. It was necessary to limit the number of
bands submitted for mass spectral analysis because of financial limitations. Each
sample costs $88 to analyze, so to test all bands would cost almost $1000. This
proved beyond the preliminary scope of this study. Remaining bands were frozen
should more funding become available.
3.3 Mass Spectrometry
The results from the mass spectrometric experiments, shown in Table 3.4,
were less than promising. A MS/MS score of at least 60, if not closer to 100 is

*
43


strongly indicative of a probable protein discovery. Two of the samples submitted
didnt have any positive matches that spanned even a single amino acid sequence.
The remaining two protein bands submitted didnt have any strong matches. Band 2
provided only one potential protein match, with relatively low confidence interval.
The identified protein, trypsin is a digestion protein solely found in the digestive
system to help digest food in the large intestine. Although this protein matches the
size of the gel band excised from the gel, and trypsin activity could help explain the
increase in motility observed with increased treatment doses; it is doubtful that
trypsin is upregulated by nitrosylation. There is no literature of aberrant nitrosylation
in the cancer phenotype. Furthermore, trypsin is used to digest the proteins within the
band, a likely source of contamination of this protein.
The results for band 4 were even less promising. Whereas at least trypsin
matched the size of the gel band excised compared to the standard ladder, none of the
potential proteins from gel band 4 came close to matching the size of the band
excised. It is believed that the long delay between completion of the gel
electrophoresis step and gel band isolation and subsequent freezing is to blame for
protein degradation and lack of detectable proteins by mass spectrometry. In addition,
the silver staining method was not the typical staining method used by the mass
spectrometry facility and involved them altering their protocol to incorporate a novel
method of destaining. This also added another variable that potentially affected the
quality of results. In the future, it would be best to choose which mass spectrometry
facility is going to be performing our analyses and ask which method of staining is
most commonly used with the highest specificity as well as any other details that
should be performed in order to optimize transfer of samples between laboratories.
f
44


Table 3.1 Excised spot potential identification
Band ID # Size (kDa) Protein Identified MS/MS Probability Score
2 26.6 Trypsin-1 precursor 19.28
3 n/a None n/a
4 510.1 Probable E3 ubiquitin-protein ligase MYCBP2 21.98
4 66 Keratin, type II cytoskeletal 1 18.58
4 565.2 Ryanodine receptor 1 16.37
4 3816.2 Titin 12.03
4 71.7 Centrosomal protein of 72 kDa 11.8
4 90.5 Protocadherin beta 1 precursor 11.41
4 146.6 TBC1 domain family member 4 11.03
8 n/a none n/a
45


4. Conclusion
4.1 Resolution of Hypothesis
The hypothesis was successfully demonstrated in regards to most of the
specific aims of the research. Synergy was observed in an overwhelming majority of
the head and neck cell lines measured strongly suggesting an epigenetic effect of
combining radiation and resveratrol. Using the biotin switch technique, it was also
shown how nitrosylation levels varied with changes in treatment groups. Nitrosylated
protein identification was not successfully determined.
4.2 Future Directions
This work could take several directions in the future in order to advance the
premises made during the course of this work. Not only were many of the
experiments left open ended, but several could also have been setup more efficiently.
Of the work that was left open ended, the easiest to finish in order to
supplement this paper are the other excised spots that have yet to be analyzed by mass
spectrometry. Due to budget restraints, only the 4 most interesting spots were
analyzed by mass spectrometric analysis in order to determine their identity. These
spots corresponded to the bands that appeared to differentiate the greatest amount
between the various treatment groups. Other bands could still be of interest based on
the fact that they are nitrosylated in head and neck cell lines potentially via treatment
with radiation but were unaffected by resveratrol or a combination therapy. However,
it could prove useful to know which proteins are reversibly denitrosylated by
resveratrol and which are unaffected by the antioxidant polyphenol.
46


Another unfinished detail of the experiment regards to better separation of
nitrosylated proteins by two-dimensional gels. Although it is certainly an option that
the biotin switch protocol that was used is simply incompatible with the isoelectric
focusing step, it seems more likely that with enough time it would be possible to
optimize the two procedures to smoothly operate together. This would allow for
better physical separation of nitrosylated proteins on gels and thus less ambiguous
identification of nitrosylated proteins. It is not immediately clear how useful it would
be to spend the time necessary researching and testing combining the biotin switch
and isoelectric focusing. The increased separation may not yield any added benefit if
the additional separation doesnt yield any novel proteins. In this case the time
contribution, which would be in the weeks to months range might be unnecessarily
lost, however if a novel protein band/spot is discovered because of the addition of
isoelectric focusing to the procedure, it would definitely justify the added time put
into developing the procedure. Unfortunately hindsight is 20:20 and not foresight.
Another area that could be improved upon in future work is the model system
in which synergy is simulated. Recently it has been observed that macrophages can
release nitric oxide in tumor microenvironments. (Wink et al., 2008) In the same
work, it was also acknowledged that in this setting nitric oxide, as is the case with
most reactive species acting in a tumor setting, acts as a double edged sword of sorts.
Nitric oxide has been shown to be used by the macrophages to induce
proinflammatory signals of the wound healing and inflammatory pathways that are
normally beneficial but also typical of tumor promotion and progression. In addition
to the beneficial effects of nitric oxide on the immune system, nitrates and nitrites
have also been found to increase cardiovascular health and fight bacterial infections.
On the contrary, nitric oxide has also been linked with tumor suppression and iNOS
expression is actually correlates inversely with tumor promotion and progression.
This demonstrates the need to continue looking at nitric oxide function in more
47


complicated in vivo models that could shine light on the nature of nitric oxide as it
functions among cooperating cell types.
As previously mentioned in the results, clonogenic assays, although the gold
standard for clinical radiation oncologists are not the most ideal method of testing
drug synergy in the lab. Should the model system prove necessary to use because of
its praise by clinical radiation oncologists, there are ways upon which it can be
improved. The change in doubling time (if any) can be researched as well as
conditions to optimize other aspects of the experimental setup such as ideal size of
wells, concentration of growth serum, length of incubation, treatment dose, and
number of doses can all be optimized.
Finally, further work on this topic would need to focus and clarify the
workings of the biotin switch protocol. Since its publication by Jaffeys group in
2001, this preferential method of detecting nitrosylated proteins has come under
heavy scrutiny for its applicability and specificity. To start, the original protocol
utilized purified proteins as a starting material instead of cell lysates to test
nitrosylation levels. Whereas this provided a clean background for research and
development purposes of reagent concentrations, etc., it left the door open for other
researchers to criticize the original methodology.
The specificity of the biotin switch methodology has been questioned on
several counts. The nitrosyl donation, blocking, ascorbate reduction and biotin
addition are all reactions that merely change the oxidation state of the cysteine that is
bound. It would be an interesting test to incubate two similarly reduced amino acids
or create protein with identical structures but replace specifically known catalytic
cysteines with tyrosine, serine or threonine amino acids that have side chain
hydroxyls that behave similar to the sulfhydryl in cysteine. This could test how
alternate side-chains react to treatment with the biotin switch protocol. One could go
48


even further and transfect these replaced proteins into cells to look at how they are
modified in vitro or in vivo derived cell lysate.
Another argument that arises is whether the nitrosylated cysteines are
physiologically relevant. Despite a change in nitrosylation with increased treatment, it
would be necessary to do three-dimensional protein modeling to determine whether
some sort of conformational changes were occurring within the protein structure or if
interactions with downstream proteins were being inhibited or promoted due to the
nitrosyl group presence. This work by a computational chemist would help validate
that the observed nitrosylation changes were physiologically relevant. The alternative
would be that the observed nitrosylation is merely a byproduct of increased oxidation
state within the cell, and that this increased oxidation is the more significant change,
rather than the resulting nitrosylation. Protein three-dimensional structure could be
affected by merely the change in oxidation and not be specific to a single residues
nitrosylation change.
49


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