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Arginine deiminase-mediated modulation of argininosuccinate and nitric oxide synthesis in cultured cell lines

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Arginine deiminase-mediated modulation of argininosuccinate and nitric oxide synthesis in cultured cell lines

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Content ARGININE DEIMINASE-MEDIATED MODULATION OF
ARGININOSUCCINATE AND NITRIC OXIDE SYNTHESIS IN
CULTURED CELL LINES
by
Wen-Chun Lin
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement of the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2003
Copyright 2003 Wen-Chun Lin
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UMI Number: 1416565
UMI
UMI Microform 1416565
Copyright 2003 by ProQuest Information and Learning Company.
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 900894695
This thesis, w ritten b y
I aJEAJ ~ C H U t i L i f t
U nder th e direction o f h.&C. Thesis
C om m ittee, and approved b y aU its m em bers,
has been p resen ted to and accepted b y The
G raduate School, in p a rtia l fu lfillm e n t o f
requirem ents fo r th e degree o f
Dean o f Graduate Studies
D ate 2003
THESIS COM M ITTEE
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DEDICATION
This thesis is dedicated to my dear parents who make my growth
full of love.
11
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ACKNOWLEDGEMENTS
This study would not have been completed without the
assistance of a number of people to whom I wish to express my
appreciation. I am deeply grateful to my advisor, Dr. Wei-Chiang
Shen, for his guidance, encouragement and care throughout my
graduate studies at USC. Also, I would like to show my
appreciation to my guidance committee, Dr. David Ann and Dr.
Curtis Okamoto, for their suggestions and comments.
I am thankful for the resources and help provided by the
staff of the Department of Pharmaceutical Sciences, USC. In
particular, I would like to thank Mrs. Daisy Shen for her dedicated
caring and patience. I appreciate all the support from my dear
friends of the 404B gang: Karin Beloussow, Adam Widera, Tinten
Lim, Jennica Zaro, Fariba Norouziyan, Rana Bahhady, Maureen
Barnes, Yun Bai, Liyun Yuan, Chris Hsu, and especially my
project cooperator, Rita Shen, for her patient technical and
knowledgeable assistance. I also realize how fortune I am to have
all the support from all of my friends all over the world.
I would also like to express my great thanks to my dear
parents, David and Vivian, and my older sister, Irene, for their
patience and for standing by me all the time. The encouragement
from my family did mean a lot to me.
iii
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TABLE OF CONTENTS
D ED ICA TIO N .......................................................................................ii
ACKNOW LEDGEMENTS................................................................. iii
LIST OF TABLES and F IG U R E S ...................................................vii
LIST OF A B B R E V IA T IO N S ..........................................................ix
A B S T R A C T .......................................................................................x
1. INTRODUCTION
1.1 Arginine and N O ................................................................. 1
1.2 Characteristics of Recombinant Arginine Deiminase
( r A D I ) ....................................................................................... 3
1.3 Argininosuccinate Synthetase ( A S ) ..................................... 6
1.4 Nitric Oxide Synthase ( N O S ) ............................................ 9
1.5 The Relationship between rADI, Argininosuccinate
Synthetase (AS) activity and Nitric Oxide (NO)
s y n t h e s i s .................................................................................10
1.6 Specific A i m s ......................................................................... 12
2. MATERIALS AND METHODS
2.1 C h e m ic a ls .................................................................................13
2.2 Cell L i n e s .................................................................................13
2.3 Cell C u ltu re.................................................................................14
2.4 Effect of ADI on cell p r o lif e r a tio n ..................................... 15
2.5 Protein A s s a y ..........................................................................16
2.6 ADI enzyme activity a s s a y :....................................................17
2.7 Arginiosuccinate Synthetase Activity Assay
2.7-1. Principle of the Enzyme and Metabolite Assays .19
2.7-2. Cell Sample P r e p a r a t i o n s .....................................20
2.7-3. Medium C o lle c t io n ........................................................20
2.7-4. Cell H arv est..................................................................21
2.7-5. Column Preparation (Resin preparation) . . .21
2.7-6. Column P acking...........................................................22
2.7-7. AS Activity A s s a y ................................................... 23
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2.8 Nitric Oxide (NO) Measurement
2.8-1. Griess Assay................................................................. 25
2.8-2. Fluorometric A s s a y ...................................................27
3. RESULTS
3.1 rADI Study
3.1-1. rADI Enzymatic Activity A s s a y ............................. 29
3.1-2. Effect of rADI on Cell Proliferation (Resistant
or Sensitive to r A D I ) ............................................30
3.2 AS Study
3.2-1. AS Activity in Different Cell Lines . . . .31
3.2-2. Influence of rADI on AS Activity in Different
Cell L i n e s ................................................................. 32
3.2-3. Influence of Cytokines on AS Activity in
Different Cell L in e s ...................................................34
3.2-4. Influence of rADI and Cytokines on AS Activity
in Different Cell L i n e s ............................................35
3.3 NO Study
3.3-1. NO in different cell l i n e s .....................................38
3.3-2. Influence of rADI on NO production . . . .39
3.3-3. Influence of cytokines on NO production . .40
3.3-4. Influence of rADI and cytokines on NO
production .......................................................... 42
4. DISCUSSION
4.1 Cells resistant to rADI have high AS activity . . .46
4.2 Induction of argininosuccinate synthetase activity by
cytokines..................................................................................... 47
4.3 rADI abolishes nitric oxide production by iNOS . . .47
4.4 Correlation between rADI, cellular argininosuccinate
synthetase (AS) activity and nitric oxide (NO) . . .49
4.5 Cytoprotective effect of arginine deiminase (rADI) . .50
4.6 Cytotoxicity caused by Tumor Necrosis Factor-alpha in
L929 50
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4.7 Further consideration
4.7-1. Selectivity of A D I ...................................................51
4.7-2. Gene expression and protein regulation . . .52
4.7-3. Localization of AS, NOS enzymes, and arginine
tr a n s p o r te r ................................................................. 52
4.8 C o n c lu sio n ................................................................................53
5. R E F E R E N C E S ......................................... 55
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LIST OF TABLES AND FIGURES
Table 1. Protein assay of L929 in two different cytokines
dosing treatments.............................................................. 34
Figure 1. Arginine and NO metabolism.........................................2
Figure 2. Several pathways of L-arginine.....................................5
Figure 3. L-arginine regeneration pathway from citrulline. .8
Figure 4. The citrulline-NO cycle...................................................9
Figure 5. The reaction catalyzed by ADI.......................................19
Figure 6. Chemical reactions in Griess method........................... 26
Figure 7. Reactions in fluorometric method used to measure
nitric oxide (NO) production.......................................... 28
Figure 8. ADI enzymatic activity....................................................29
Figure 9. The effect of rADI on cell proliferation. . . .31
Figure 10. AS activity in cells without any treatment. . . .32
Figure 11. AS activity in cells treated with rADI 1 mU/ml for
24 hours...............................................................................33
Figure 12. AS activity in cells treated with Interferon-gamma
(IFN-y) 50 unit/ml and Tumor Necrosis
Factor-alpha (TNF-a) 5 ng/ml for 24 hours. . .35
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Figure 13. AS activity in cells treated with Interferon-gamma
(IFN-y) 50 unit/ml and Tumor Necrosis
Factor-alpha (TNF-a) 5 ng/ml and rADI 1 mU/ml
each alone or together for 24 hours...............................37
Figure 14. AS activity in L929 cells treated with different
dosages................................................................................ 38
Figure 15. Nitric oxide (NO) production in cells without any
treatment..............................................................................39
Figure 16. NO production in cells treated with rADI 1 mU/ml
for 24 hours.........................................................................40
Figure 17. NO production in cells treated with
Interferon-gamma (IFN-y) 50 unit/ml and Tumor
Necrosis Factor-alpha (TNF-a) 5 ng/ml for 24
hours.................................................................................... 42
Figure 18. NO production in cells treated with
Interferon-gamma (IFN-y) 50 unit/ml and Tumor
Necrosis Factor-alpha (TNF-a) 5 ng/ml and rADI
1 mU/ml each alone or together for 24 hours. . .44
Figure 19. NO production in L929 cells treated with different
dosages................................................................................ 45
v iii
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LIST OF ABBREVIATIONS
ADI: arginine deiminase
rADI: recombinant ADI
AS: argininosuccinate synthetase
AL: argininosuccinate lyase
NO: nitric oxide
NOS: nitric oxide synthase
iNOS: inducible NOS
eNOS: endothelial NOS
TNF-a: tumor necrosis factor alpha
IFN-y: interferon gamma
DMEM: Dulbecco’s modification of Eagle’s Medium
MEM: minimum essential medium
MEM a+: Dulbecco’s minimum essential medium alpha
PBS: phophate-buffered saline
FBS: fetal bovine serum
L-arg: L-arginine
L-cit: L-citrulline
MDCK: Madin-Darby canine kidney
CHO: Chinese hamster ovarian
A549: human lung carcinoma
HeLa: human cervix adenocarcinoma
MCF-7: human mammary adenocarcinoma
L929: mouse fibroblast
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ABSTRACT
Arginine deiminase (ADI), an amino acid degrading
enzyme, was found to have the potential to become a novel
therapeutic drug for the treatment of cancer and some diseases
caused by the over-production of nitric oxide. Arginine deiminase
(ADI) is a Mycoplasma enzyme that catalyzes the deimination of
L-arginine to L-citrulline and ammonia. However, the detailed
mechanism of ADI on cells is still not clear. The correlation
between the cell proliferation rate and rADI and the
argininosuccinate synthetase (AS) activity in six cell lines
(MDCK, CHO, A549, HeLa, MCF-7, and L929) was investigated.
Also, the effects of rADI and cytokines on argininosuccinate
synthetase (AS) activity and nitric oxide (NO) production were
determined. The probable mechanisms of rADI in modulation of
AS activity and NO production are discussed.
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1. Introduction
Using amino acid degrading enzymes was thought to be one
treatment solution in cancer therapy. However, L-asparaginase is
the only one used clinically treat acute lymphoblastic leukemia
and few sub-types of non-Hodgkin’s lymphoma [Wu and Morris,
1998]. Besides L-asparaginase, the biochemistry and physiology of
L-arginine have to be reconsidered as the brightness of the recent
research that L-arginine is the only substrate o f polyamine
synthesis, as well as all isoforms of nitric oxide synthase (NOS)
[Muller and Boos, 1998]. NOS is the enzyme regulates nitric oxide
(NO), which functions significantly in life. Also, deprivation
studies have demonstrated the unique requirement of L-arginine
for cultured transformed and malignant cells as well as human
diploid fibroblasts [Scott et al., 2000; Wheatley et al., 2000].
Therefore, L-arginine, as a unique and versatile amino acid, would
seem to be an ideal candidate for therapeutic enzymatic amino acid
degradation [Shen et al., 2003].
1.1 Arginine and NO
Cellular NO production depends on the availability of
arginine, a substrate for NOS. Arginine is transported from
extra-cellular spaces via the cationic amino acid transporter (CAT)
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and regenerated from citrulline, a product of NOS reaction, by the
citrulline-NO cycle which consists of NOS, argininosuccinate
synthetase (AS) and argininosuccinate lyase (AL) (fig.l) [Mori and
Gotoh, 2000; Wu and Morris, 1998; Wu and Brosnan, 1992]. Previous
research findings indicate that citrulline-arginine recycling is
important for NO production. [Koga et al., 2003], [Flodstrom et al.,
1996; Simmons et al., 1996], [Zhang et al., 2000].
Glutamate Proline
\ ^
P5C Polyamine
/ O D C
Urea ^ Ornithine
Arginase
U l
A rginm e-jc/vr j-*-Arginine
NOS
Fumarate
^-Citrulline
AS Aspartate
Argininosuccinate
Fig 1. Arginine and NO metabolism. The lower triangle shows the
citrulline-NO cycle composed of NO synthase (NOS),
argininosuccinate synthetase (AS) and argininosuccinate lyase
(AL). CAT, cationic amino acid transporter; OAT, ornithine
aminotransferase; ODC, ornithine decarboxylase; P5C,
pyrroline-5-carboxylate [Koga et al., 2003].
2
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Arginine is a substrate of all isoforms of NO synthase and
generates L-citrulline from L-arginine and molecular oxygen NO
via a five-electron transfer reaction. Although the NOS enzymes
can metabolize both intracellular and extracellular arginine, it has
been suggested that it is primarily the extracellular arginine that is
metabolized into NO, after it is transported inside the side the cell
via a cationic amino acid transporter [Dillon et al., 2002; McDonald
et al., 1997]. Moreover, it seems that arginine is the only
physiological substrate for the generation of NO in eucaryotic cells,
and arginine analogues are potent inhibitors of NOS [Grant et al.,
1998]. Thus the pathophysiological roles of arginine are
intertwined with the biological effects of NO [Morris et al., 1999],
and many functions of NO have been delineated from the
attenuation or enhancement of the respective effect in the presence
of arginine-based NOS inhibitors. Several aspects of the
modulation of NO synthesis by the availability o f arginine have
been under investigation [Wiesinger, 2001].
1.2 Characteristics o f Recombinant Arginine Deiminase (rADI)
ADI was cloned by the polymerase chain reaction (PCR)
from the mycoplasma genomic DNA [Mieawa et al., 1994] and the
recombinant ADI (rADI) was purified to homogeneity in our
laboratory [Beloussow et al., 2002].
3
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Arginine deiminase (ADI) is a Mycoplasma enzyme that
catalyzes the deimination of L-arginine to L-citrulline and
produces ammonia as a by-product (fig.2). It has been reported
that ADI can inhibit cell proliferation in vitro [Ensor et al., 2002;
Gong et al., 2000; Gong et al., 1999; Miyazaki et al., 1990; Takaku et
al., 1995] and tumor growth in vivo [Ensor et al., 2002; Takaku et al.,
1992]. The anti-tumor activity of ADI was observed in
tumor-bearing mice with no apparent toxicity at 100 times the
minimum effective dose [Takaku et al., 1992]. The mechanism by
which ADI exerts its anti-tumor effect has yet to be determined;
however, it has been suggested that it may be due to simple amino
acid depletion or inhibition of tumor angiogenesis [Beloussow et
al., 2002; Gong et al., 1999].
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citrulline + NO
citrulline N-hydroxy-L-arginine
NOS's
Arginase
L-Arginine
urea + ornithine
ADI
Polya mines citrulline + NH3
apoptosis
angiogenesis
Angiogenesis
Proliferation
Fig 2. Several pathways of L-arginine. ADI hydrolyzes L-arginine
to form L-citrulline and ammonia. ( ^ ) are the enzymes
involved in the pathways. | | are the biological effects of the
regulation.
ADI appears to possess a number of biochemical properties.
First, this enzyme retains much of its enzymatic activity at
physiological pH. Second, this enzyme has a high affinity for
arginine (Km -30 pM) and thus is able to lower extracellular
arginine levels to <5 pM [Dillon et al., 2002].
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1.3 Argininosuccinate Synthetase (AS)
Argininosuccinate synthetase catalyzes the reversible
conversion of citrulline, aspartate, and ATP to argininosuccinate,
AMP, and inorganic pyrophosphate. There are three important
metabolic processes that require this process. First, in all
organisms AS catalyzes the rate-limiting step in the biosynthesis
of arginine, one of the 20 natural amino acids, and a precursor for
the synthesis of several other bio-molecules.
Second, AS participates in the urea cycle, a five-enzyme
cycle that employs four of the enzymes of arginine biosynthesis to
detoxify ammonia through the production of urea. Ammonia
detoxification is critical for the survival of higher organisms. In
humans, failure to produce functional AS leads to the buildup of
citrulline, ammonia, and orotic acid. If untreated, the neurotoxic
ammonia can cause brain damage and coma, and in cases where
urea cycle function is significantly compromised (less than 5%
activity) the condition is typically fatal [Lemke and Howell, 2002}.
Finally, AS and a second urea cycle enzyme,
argininosuccinate lyase, together with the flavoprotein nitric-oxide
synthase form the arginine-citrulline cycle, an abbreviated urea
cycle that provides de novo arginine biosynthesis for sustainable
overproduction of nitric oxide (NO) [Xie et al., 2000]. NO is a small,
membrane-permeable, highly reactive molecule that plays key
6
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roles in a wide range of mammalian processes including blood
pressure control, neurotransmission, apoptosis, immune system
function, and wound healing [Akaike and Maeda, 2000; Karupiah et
al., 2000; Kroncke et al., 2000; Melino et al., 2000], Over-expression
of AS for sustained nitric oxide production via the
arginine-citrulline cycle leads to the potentially fatal hypotension
associated with septic and cytokine-induced circulatory shock. AS
is the rate-limiting enzyme in both the urea and the
arginine-citrulline cycles [Xie and Gross, 1997] and is therefore a
key participant in all these pathways [Lemke and Howell, 2002],
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angiogenesis
*
citrulline + NO
t
citrulline N-hydroxy-L-arginine
r
Arginase
urea + ornithine + -L ------- L - A r g in in e <
i
Polyamines citrulline + NH 3
\ | / ______
Proliferation
&
Angiogenesis
apoptosis
NOS s
re g e n e ra tio n pathw ay
o f L -a rg in in e fro m
c itru llin e
citrulline
argininosuccinate synthase"
*
rgininosuccinate lyase.
Fig 3. L-arginine regeneration pathway from citrulline.
Argininosuccinate synthase (AS) and argininosuccinate lyase (AL)
are involved in the pathway of arginine regeneration from
citrulline. C~^) are the enzymes involved in the pathways.
I I are the biological effects of the regulation.
8
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■NO
A s p a rta te
u-Keto
. acid
/ NOS
\ [ A m m o
A rginine
*
Km 3.0mM \
\ / Km 0.2mM
ar.;d
A rgm ino
Succinate
O x a to a c e ta te
/
/ \
' - 4
Fumarale M a la te
Km 5.3mM
Fig 4. The citrulline-NO cycle. NOS can be viewed as a
component of a citrulline-NO cycle whereby transamination
reactions feed nitrogen atoms onto a carrier molecule to generate
arginine, which ultimately provides the nitrogen atom for NO
[Nussler et al., 1994].
1.4 Nitric Oxide Synthase (NOS)
Nitric oxide (NO), a simple diatomic free radical, has
multiple activities under physiological and pathological conditions
acting as a natural anticoagulant, vasodilator, neuro-transmitter,
and mediator of immune system function [Abramson et al., 2001;
Alderton et al., 2001; Bogdan, 2001].
NO is synthesized from L-arginine by the catalytic action
of a group of enzymes termed nitric oxide synthase (NOS)
[Alderton et al., 2001; Davis et al., 2001; Mori and Gotoh, 2000]. At
least 3 different NOS isozymes exist. Two of which are
constitutively expressed including neuronal NOS (nNOS) and
endothelial NOS (eNOS), and one of which is inducible (iNOS).
9
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NO can be made in much larger quantities by iNOS. Inducible
nitric oxide synthase (iNOS or NOSH) is a major enzyme
regulating the inflammatory process in macrophages and
endothelial cells [Balligand et al., 1995; Mori and Gotoh, 20001. iNOS
synthesis is increased dramatically in response to endotoxin and
pro-inflammatory cytokines. It has been suggested that NO
synthesis may also play a protective role in inflammation and aid
in the killing of infectious organisms [Akaike and Maeda, 2000;
Bogdan, 2001; Brunet, 2001; Colasanti and Suzuki, 2000]. However,
overproduction of NO has also been associated with numerous
pathological conditions including stroke, diabetes mellitus and
demyelinating disorders such as multiple sclerosis [Dillon et al.,
2002], [Borderie et al., 1999; Du etal., 2002; Kwon et al., 2001; Lee et
al., 2001; Neufeld et al., 1999; Piepot et al., 2002; Yang et al., 1994].
Therefore, suppression of NO synthesis may be an important target
for the treatment or prevention of these diseases. It is crucial to
understand the mechanisms underlying its production.
1.5 The Relationship between rADI, Argininosuccinate
Synthetase (AS) activity and Nitric Oxide (NO) synthesis
Argininosuccinate synthetase (AS) and nitric oxide
synthase (NOS) comprise part of the cyclic metabolic pathway to
produce nitric oxide (NO). AS is one of the arginine synthesis
10
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enzymes synthesize argininosuccinate from aspartate and
citrulline. NOS forms NO and citrulline from arginine. With the
discovery of arginine-derived nitric oxide (NO) as a key cell
signaling molecule [Nathan, 1992; Stuehr and Griffith, 1992], the
possibility arises that the function of AS and AL is to convert
citrulline, a co-product of nitric oxide synthase (NOS), back to
arginine for continued NO production. Previous research [Mitchell
et al., 1990; Wu and Brosnan, 1992] showed that cytokine-activated
cells, which produce large quantities of NO, have an increased
capacity to produce arginine from citrulline. Thus, up-regulation
of AS activity could function to regenerate arginine as substrate
for the inducible isoform of NOS (iNOS). Evidence also showed
that imminostumulant-induced AS plays a key role in NO
formation by vascular cells when arginine becomes limiting
[Hattori et al., 1994].
On the other hand, ADI, the potential drug for anti-cancer
treatment, can consume arginine to citrulline, and thus, interfere
the citrulline-NO cycle. The question is how rADI regulates NO
production? How it affects the AS activity in the
arginine-citrulline cycle? The answers to the questions would be
helpful to reveal the mechanism of ADI.
11
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1 .6 Specific Aims
The correlation of cell proliferation and argininosuccinate
synthetase (AS) activity in six cell lines was investigated. Also,
the effects of rADI and cytokines on argininosuccinate synthetase
(AS) activity and nitric oxide (NO) production were demonstrated.
The possible mechanism of rADI-modulation of AS activity and
NO production are discussed.
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2. Materials and Methods
2.1 Chemicals
Chemicals and reagents were obtained from Sigma
Chemical Company (St. Louis, MO) unless otherwise stated. Fetal
bovine serum, trypsin-EDTA, penicillin/streptomycin, powdered
culture media and other cell culture products were purchased from
Gibco. Dowex 1-X8-200-400 resin was obtained from Supelco
(Bellefonte, PA). Micro BCA protein assay reagent kit was
obtained from Pierce (Rockford, IL). Rat recombinant IFN-y,
TNF-a, nitrite standard and Griess Reagent were purchased were
from Calbiochem (La Jolla, CA). [1 4 C] Aspartic acid (200
mCi/mmol) was purchased from Moravek Biochemicals (Brea,
CA). ADI was cloned by PCR from Mycoplasma genomic DNA
and the recombinant ADI was purified to homogeneity in our
laboratory [Beloussow et al., 2002]. The specific activity of the
renatured rADI was 32.7 U/mg protein, similar to the reported
value for mycoplasmic-derived ADI (37 U/mg).
2.2 Cell Lines
The following cell lines were all obtained from American
Type Culture Cell (ATCC) (Manassas, VA) and maintained in
medium recommended by ATCC. Mouse fibroblast (L929) was
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maintained in minimum essential medium (MEM). Human
mammary adenocarcinoma (MCF-7) was maintained in Dulbeccos
Modification of Eagles Medium (DMEM). Human cervix
adenocarcinoma (HeLa) was maintained in minimum essential
medium (MEM). Human lung carcinoma (A549) was maintained
in Dulbeccos Modification of Eagles Medium (DMEM). Chinese
hamster ovarian (CHO) was maintained in alpha + minimum
essential medium (MEM a+). Madin-Darby canine kidney
(MDCK) was maintained in minimum essential medium (MEM).
All of them were supplemented with 10 % fetal bovine serum and
0.5 % of penicillin/streptomycin.
2.3 Cell Culture
All cell cultures were routinely maintained at 37°C in 25
cm2 cell culture flasks in an atmosphere of 95% air and 5% C 0 2 in
a humidified incubator. Appropriate cell culture media and PBS
solution were prepared and sterilized in our laboratory following
the manufacturers’ directions. All cell lines grew as monolayers.
All cell stocks (seeded at 50,000 cells for 25 cm2 culture flask)
were maintained in the appropriate medium supplemented with
penicillin and 10% FBS (by volume). The cells were passed when
they reached confluence on approximately day 5 (ascertained by
light microscopy). At confluence, the culture medium was
14
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removed and two times of 5 ml PBS washing was followed. The
stock cells were detached from the culture flask by trypsinization
(0.25 ml 10X trypsin/EDTA) for 3 minutes at 37°C. Fresh culture
medium, warmed to 37°C, was then added, and a single cell
suspension was achieved by drawing up and suspending by pipette
and releasing the contents of the pipette against the inside wall of
the culture flask thereby “crushing” or separating the cell
aggregates. MDCK, required extra pipetting of the cell suspension
up and down 5 additional times by using 18G 1 1/2 needle from
Becton Dickinson (B-D) attached to a 5 ml syringe in order to
avoid cell aggregation when passing the cells. To assure single
cell suspension, the flask was checked by light microscopy. The
cell suspension was counted in a Coulter Model ZF counter
(Coulter Electronics, Hialeah, FL) and appropriate dilutions made
for seeding new stock flasks or cluster well plates for
experimentation.
2.4 Effect o f AD I on cell proliferation
The effect of the arginine deiminase (ADI) on cell
proliferation was assessed in the various cell lines. The test cells
(around 6000 cells per well) were plated into 24-well culture
plates in 1 ml of respective media (DMEM was used for MCF-7
and A549. MEM was used for L929, HeLa, and MDCK. MEM a+
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was used for CHO.), supplemented with penicillin/streptomycin
and FBS. Cells were treated with various rADI concentrations
from 0, 0.01, 0.03, 0.1, 0.3, and 1.0 mU/ml in triplicate wells.
After 4-day incubation at 37°C in 95% air/5%C02 humidified
incubator, cells were rinsed with cold PBS and protein content in
each well was determined by using Pierce Protein Assay. The data
are presented as percent of protein content in the control wells.
2.5 Protein Assay
Pierce Assay based on the colorimetric determination by
using bicinchoninic acid [Smith et al., 1985]. A BSA standard (0,
10, 40, 80, 100 ng/ml) was used each time for quantifying results.
Inhibition of cell growth is expressed as % control. After
removing the culture medium from each well and washing two
times each with 1 ml cold PBS per well in a 24-well plate, 1 ml 1
N NaOH was then added to each well to lyse the cells. The plate
was then incubated at 37°C for 10 minutes. After mixing the
NaOH solution of each well with a Pasteur pipette, a 100 pi
aliquot of the sample, in duplicate, was place in a 96-well plate
for the Pierce assay. The Microtiter Plate Protocol for the Pierce
Micro BCA Protein Assay Kit is: the working reagent was
prepared by combining 50 parts Micro Reagent A (containing
sodium carbonate, sodium bicarbonate and sodium tartrate in 0.2
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
N NaOH), 48 parts Micro Reagent B (containing 4% BCA in
water), and 2 parts Micro Reagent C (containing 4% cupric sulfate,
pentahydrate in water). Then 100 pi of the Micro Reagent mixture
was added to each microtiter plate well which already contained a
100 pi of a standard, 1 N NaOH (as solvent background control),
or unknown concentration samples. All the solution was mixed
gently in the plate then covered and incubated at 37°C for 2 hours.
The absorbance was then read at 570 nm with a microtiter plate
reader. To obtain a protein concentration, a standard curve was
plotted and corrected at 570 nm vs. the protein concentration.
Inhibition of the cell growth was calculated as the average of the
absorbance for the treatment wells over the average absorbance of
the control wells. Note: the low concentration of NaOH used to
lyse the cells was found not to interfere in the protein
determination, data not shown.
2.6 A D I enzyme activity assay:
The ADI activity was determined by the amount of
L-citrulline produced from L-arginine. L-citrulline concentration
from 0 to 400 pM was used for determining the standard curve
since ADI can convert L-arginine to L-citrulline. 100 pi of
lOOmM arginine was added to 5 tubes. 10 pi of ADI was diluted
with 100 pi of PBS, then 15pi of ADI dilution was added to the 5
17
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
tubes and incubated in 37°C water bath for various time periods
(0, 5, 10, 15, and 20 minutes). Reaction was terminated by adding
lOOpl of a 1:3 mixture (v/v) of concentrated H2SO4 and
concentrated H 3PO 4 solution and 60p,l of 2,3-butanedione
monoxime were added to the 5 tubes. Both terminating solutions
were added to L-citrulline standard solution, as well. After mixing
each tube thoroughly by vortexing, they were incubated at 90°C
for 30 minutes. They were the allowed to the room temperature
with occasional shaking. Afterwards, the absorbance of the orange
color developed by the reaction product of L-citrulline and
2,3-butanedione monoxime was measured at wavelength 490nm
by a spectrophotometer. The amount of citrulline quantified by
comparison with a standard curve prepared with L-citrulline. One
unit (U) of ADI activity is defined as the amount of enzyme that
converted 1 pmol of L-arginine to L-citrulline per minute under
assay conditions. Arginine, by itself, does not give a color
reaction and thus causes no interference.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
h 2 o n h 3
c h 2 c h 2
c h 2
c h 2
ADI
CH2
c h 2
H n h3 + H n h 3 +
COO" COO"
Arginine Citrulline
Fig 5. The reaction catalyzed by ADI.
2.7 Arginiosuccinate Synthetase Activity Assay
2.7-1. Principle of the Enzyme and Metabolite Assays
Arginiosuccinate synthetase activity in cell homogenates
was determined by measuring the conversion of [14C] aspartate
and citrulline into [14C] arginigosuccinic acid [Hattori et al., 1994;
Jackson et al., 1996; Nussler et al., 1994; O'Brien, 1979; O'Brien and
Barr, 1981]. Protein concentration was determined by a
commercially available assay kit. AS enzyme specific activities
are expressed as pmol argininosuccinate formed from citrulline
per minute per mg protein.
1 9
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2.7-2. Cell Sample Preparations
Each cell line was seeded in eight T75 flasks with around
20% confluence in the medium assigned in different cell lines at
day 0. Each treatment group would be duplicated. On day 2, all
the media were changed to DMEM/F12 phenol-red free media in
order to avoid interference with the NO fluorometric assay. The
cells were dosed according to the different treatment groups:
control, cytokine, rADI, and cytokine plus rADI. Interferon
gamma (IFN-y) 50 unit/ml plus tumor necrosis factor alpha
(TNF-a) 5 ng/ml were used in the cytokine treatment groups,
except for L929 cells, where TNF-a 2.5 ng/ml instead of 5 ng/ml
was used. L929 are very sensitive to TNF-a, and a higher dose of
TNF-a would cause cytotoxicity. The concentration of 1 mU/ml of
rADI was used. In the cytokine plus rADI group, 1 mU/ml of
rADI plus IFN-y 50 unit/ml plus TNF-a 5 ng/ml were used. After
incubation in a 37°C incubator for 24 hours, cells were harvested
on day 3.
2.7-3. Medium Collection
On day 3, one ml of medium was collected from each T75
flask. The medium was centrifuged at 1500 rpm for 5 minutes to
remove cell debris. 150 pi of the supernatant was used for NO
20
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fluorometric assay, and another 100 pi of the supernatant was
used for NO Griess assay.
2.7-4. Cell Harvest
Cells were harvested from the T75 flask by removing the
medium, then washed twice with 10 ml of ice-cold PBS and
scraped with a Teflon policeman, then an additional 7 ml of iced
PBS was added to each T75 flask. Cell suspensions of the each
treatment group for each cell line were added together. Cells
suspensions were then centrifuged at 1000 rpm for 10 minutes.
The supernatant was removed and discarded and 500 pi of lOmM
Tris-HCl, pH 7.5 was added to lyse the pellets and then tapping
the pellets slightly to re-suspend into solution. This cell
suspension was frozen in dry ice with an acetone solution and
thawed in a 37°C water bath three times. Lysates were centrifuged
at 12,000 g (4600 rpm) for 20 minutes. The supernatant fluid was
harvested as the cell extract for both the following AS activity
assay and the protein assay.
2.7-5. Column Preparation (Resin preparation)
Dowex lX 8-acetate was prepared from Dowex 1X8-C1
(200-400 mesh) Resin. Sufficient distilled-deionized water
(dd-water) was added to cover the resin bed in a 500 ml beaker
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and the resin was stirred gently for one minute to ensure complete
mixing. The resin was allowed to stand for 15 minutes, and the
water carefully decanted. Fresh dd-water replaced and the
procedure repeated once again, but for 5 to 10 minutes. The water
was discarded and the resin was re-suspended in 3 M sodium
acetate for 48 hours with slight stirring. The resin was then
equilibrated with 0.05 M acetic acid. The equilibrated solution
was then degassed to prevent bubbles from forming in the column
preparation.
2.7-6. Column
One ml B-D syringe was used as the column instead of 0.5
X 4 cm columns in the reference paper [Jackson et al., 1996]. Glass
fiber filter (Reeve Angel type, grade 934 AH, NJ) from Whatman
was cut by three-hole puncher into a circle with 0.7 cm in
diameter. The filter was pushed all the way down to the neck of
the syringe by a long glass Pasteur pipette. B-D 18G 1 1/2 needle
was connected to the syringe in order to increase the flow rate.
The column was placed vertically to form a bed, and the resin
with 0.05 M acetic acid solution was applied to the column by
glass Pasteur pipette. The resin was up to 0.7 ml scale. The pH
value of the effluent was checked by pH meter.
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.7-7. AS Activity Assay
200 (il of the cell supernatant fluid was applied to 12 X 75
mm glass tube. Asparate was at a concentration of 30 pM (0.021
pCi/nmol). Reaction mixtures also contained L-citrulline (5 mM),
Tris-HCl (10 mM, pH 7.5), ATP (0.1 mM), Phosphoenolpyruvate
(1.5 mM), Pyruvate kinase (4.5 units), myokinase (4 units), and
pyrophosphatase (0.2 units) in a total volume of 0.3 ml. In another
words, each 12 X 75 mm glass tube contains 100 pi of the
following reagents and 200 pi of the cell supernatant:
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reagents Final
conc.
Stock conc. Volume added
Aspartic acid
30 pM 500 pM
18 pi (1.8 pi hot
+ 16.2 pi cold)
Citrulline 5mM 60 mM 25 pi
ATP 0.1 mM 20 mM dilute to
1.5mM 20 pi
Phosphoenolpyruvate 1.5mM 75 mM dilute to
37.5 mM 12 pi
Pyruvate kinase 4.5 unit 0.9 unit/pl 5 pi
Myokinase 4 unit 0.8 unit/pl 5 pi
Pyrophosphatase
0.2 unit
0.1 unit/pl dilute
to
0.04 unit/pl 5 pi
MgC12 6mM 360 mM 5 pi
KC1 20mM 1.2 M 5 pi
* Tris-HCl 10 mM (pH 7.5) was used for all the dilutions.
Two glass tubes of 200 pi dd-water instead of the cell
supernatant were used as the background control group. Reactions
were allowed to proceed in 12 X 75 mm glass tubes at 37°C for 90
minutes. At the end of the incubation period, 0.05 ml of 1 M
acetic acid was added and the tubes were heated to 90°C for 30
minutes. 0.65 ml of dd-water was added at the end of the
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
incubation. Using ion exchanged chromatography on Dowex 1-X8
(200-400 mesh), the respective substrates was separated from the
[14C] argininosuccinate product, then quantified by liquid
scintillation counting. The mixture with the total volume of 1 ml
was applied to the syringe columns of Dowex 1-X8 acetate
equilibrated with 0.05 M acetic acid. One ml of 0.05 M acetic acid
was used to rinse the reaction column and also applied to the
column twice. The entire 3 ml effluent was collected from the
column and mixed well. One ml of the effluent was taken out and
added to 5 ml of Aquasol for scintillation counting to determine
the radioactivity.
2.8 Nitric Oxide (NO) Measurement
2.8-1. Griess Assay
NO is rapidly converted into nitrate and nitrite in aqueous
solutions. Since nitrite is the major product of NO in tissue
culture, the Griess method can be used for determining NO
concentration, and is the simplest and most convenient method to
measure nitrite in aqueous solutions. Sodium nitrite (NaN02)
standard solution and assay buffer from Cayman Chemical
Company (Ann Arbor, MI) werfe used to make the nitrite standard
solutions: 5, 10, 15, and 20 pM. Cell-free culture supernatants
were collected (100 pi), mixed with 50 pi of Griess Reagent 1
25
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(containing 0.33% sulfanilamide in 15% acetic acid) and 50 pi of
Griess Reagent 2 (containing 0.133% N-l-naphthyl
ethylendiamine) in a 96-well plate. After leaving for 10 minutes at
room temperature, the plate was measured at an absorbance of
570 nm by a spectrophotometer (Dynatech MR 700). Nitrite
concentrations were quantified by comparison with the standard
curve. This method can be used to accurately measure as little as
ljiM nitrite in aqueous solutions.
Fig 6 . Chemical reactions in Griess method [Nims et al., 1996].
N
III
S O ,N H 2
Sulfanilamide
[Griess Reagent 1]
-IMH,
N-( 1-Naphthyl)
echylenedianiine
[Griess Reagent 2]
N
s o 2nh._.
Azo product
540 nm
26
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2.8-2. Fluorometric Assay
The amount of nitric oxide was determined by
N-nitrosation of 2,3-diaminonaphthalene (DAN) to yield
2,3-naphthotriazole (NAT) by NO-derived N-Nitrosating agent
(NO X)[M iles et al., 1996; Misko et al., 1993], Nitrite standard
solution was freshly prepared from N aN 02 powder (M.W.= 69), to
make 0, 10, 100, 250, 500, 1000, and 2000 pM. 150 pi of the
sample supernatant as well as each standard solution was prepared
in a 1.5 ml eppendorf, 15 pi of DAN (0.05 mg/ml in 0.62 M HC1)
was added and mixed immediately, then followed by ten minutes
of incubation at room temperature. 7.5 pi 2.8 M NaOH was then
added, then vortexed immediately to terminate the reaction. The
time of the reaction is crucial, so exactly the same incubation time
for each sample was ensured. Total of 150pl of the mixture was
then transferred to a black 96-well plate from Thermo Labsystems
(Franklin, MA). Fluorescence measurements were read using a
TEC AN Fluorostate Microplate Reader, with excitation at 360 nm
and emission at 430 nm. Note: this method is based on the
measurement of fluorescence emission, so keeping the reaction in
a dark environment is important. This fluorometric method is more
sensitive than the Griess method. The fluorometric assay can
detect as little as 10 to 30 nM nitrite in the final reaction mixture.
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2,3-Diaminonaphthalene (DAN)
N=N-OH
2,3-Naphthotriazole (NAT)
N-Nitrosation of 2,3-diaminonaphthalene (DAN) to yield 2,3-naphthotriazole (NAT) by
NO-derived N-Nitrosating agnet (NOX).
Fig 7. Reactions in fluorometric method used to measure nitric
oxide (NO) production [Miles et al., 1996].
28
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3. Results
3.1 rADI Study
3.1-1. rADI Enzymatic Activity Assay
The enzymatic activity of rADI was determined prior to
each experiment. The amount of citrulline produced from arginine
was linear in the correlation to the incubation time, indicating that
rADI converted the L-arginine to L-citrulline steadily. The rADI
enzymatic activity was then determined as 32.7 U/mg, where 1
unit is defined as 1 pmole/min.
■ § 4000
J. 3500
H 3000
O
■ a 2500
0
a - 2000
v
1 1500
.1 1000
o
]5 500
o
0
incubation time (min)
Fig 8. ADI enzymatic activity. The amount of substrate,
L-arginine, was transformed into L-citrulline due to the ADI
enzymatic activity versus the different incubation time.
29
0 5 10 20 25 15
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3.1-2. Effect of rADI on Cell Proliferation (Resistant or
Sensitive to rADI)
Experiments were performed to determine the range of
rADI concentrations that would elicit growth inhibition. The in
vitro growth-inhibitory activity of rADI in different cell lines is
shown in Fig 9. rADI strangly inhibited the growth of CHO and
MDCK. The rADI concentration required for 50% growth
inhibition (IC50) was below 0.1 mU/ml for CHO and MDCK.
A549 cell proliferation occurred in a dose-response manner at
concentrations from 0.01 mU/ml and 1.0 mU/ml, the IC50 value
for A549 was 0.2 mU/ml. No significant inhibition was observed
in the remaining three cell lines, L929, MCF-7, and HeLa, even at
1.0 mU/ml rADI, which was the highest concentration that was
tested. The rADI concentration used in all further rADI
experiments was 1 mU/ml.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
120 i
— L929
— MCF7
— A—HeLa
-X -A 5 4 9
-O -C H O
-i2r-M DCK
iooy
o
J 6 0 -
40 -
20 -
0.01 0.1 1
rADI (U/L)
Fig 9. The effect of rADI on cell proliferation. Cells were seeded
in 24-well culture plates in respective media and supplements and
treated with rADI at the indicated concentrations. Cells were
incubated at 37°C, 95% air/5%CC> 2 in the presence of rADI until
control cells (without rADI) reached confluence. The cells were
then assayed for protein content as a measurement of cell number
and expressed as % control ± SD, n=3.
3.2 AS Study
3.2-1. AS Activity in Different Cell Lines
The AS activities in the cell homogenates of the six cell
lines ranged from undetectable to 111.6 pmol/min per mg protein
and are shown in Fig 10. Among the tested cell lines, MDCK and
CHO, the most susceptible to rADI treatment, exhibited the
lowest AS activity (-1.4 ± 0.2 and -2.8 ± 0.4 pmol/min per mg).
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A549, moderately susceptible to rADI treatment, exhibited AS
activity of 18 ± 1.2 pmol/min/mg. HeLa, on the other hand,
resistant to rADI treatment, exhibited low AS activity, 0.1± 0.1
pmol/min/mg. Moreover, both L929 and MCF-7 which were
resistant to rADI treatment showed the higher AS activities i.e.,
27.5 ±0.8 and 111.6 ± 1.2 pmol/min per mg respectively.
1 20
1 0 0
C 5
E
80
"c
£
o 60
E
a.
>.
40
>
o
C O
20
C O
<
0
-20
MDCK CHO A549 HeLa MCF-7 L929
Fig 10. AS activity in cells without any treatment. The AS activity
was measured when the cells reached confluence after 3 days.
3.2-2. Influence of rADI on AS Activity in Different Cell
Lines
Cells were treated with 1 mU/ml rADI for 24 hours and
AS activities were measured in order to see the effect of rADI on
AS activity. The data showed diverse effects of rADI on AS
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
activities in the six cell lines. For most of the cell lines, CHO,
A549, MCF-7, and L929, showed a decreased AS activity (-8.2 ±
2.9, 5.8 ± 1.9, 55.9 ± 2.4, and 11.5 ± 1.0 pmol/ml/mg). Only
MDCK and HeLa exhibited increased AS activities. This increase
was small in MDCK (-0.9 ± 0.1), and overall AS activity in
MDCK was low. However, a large increase in AS activity was
observed in HeLa cells. The AS activity in HeLa cells increased
from 0.1 ± 0.1 to 62.6 ± 4.5 pmol/min/mg with rADI treatment.
120
□ Control
EA D I
100
80
60
40
20
0
MDCK A549 HeLa MCF-7 L929
-20
Fig 11. AS activity in cells treated with rADI 1 mU/ml for 24
hours. The results were compared with the control groups without
any treatment. CHO, A549, MCF-7, and L929 all showed
decreased AS activity; while MDCK and HeLa cells exhibited an
increase in AS activity.
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.2-3. Influence of Cytokines on AS Activity in Different
Cell Lines
The effects of cytokines on AS activity was also
investigated. Interferon-gamma (IFN-y) 50 unit/ml and Tumor
Necrosis Factor-alpha (TNF-a) 5 ng/ml were used. However, due
to the severe cytotoxicity of TNF-a 5 ng/ml in L929, a lower dose
of TNF-a 2.5 ng/ml was used.
L929 Total Protein Amount (mg)
control 7.19
ADI 7.70
cytokine (5ng/ml TNF- a ) 0.66
cytokine + ADI 0.98
control 7.95
ADI 8.77
cytokine (2.5ng/ml TNF- a ) 2.53
cytokine + ADI 4.08
Table 1. Protein assay of L929 in two different cytokine dosing
treatments. The dose of Interferon-gamma (IFN-y) 50 unit/ml and
Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml were used in the
upper panel; while the lower dose of TNF-a 2.5 ng/ml was used in
the bottom panel.
MDCK, CHO, and A549 all showed similar levels of the
AS activities (0.0 ±0.1 pmol/min/mg in MDCK, -2.8 ± 0.1
pmol/min/mg in CHO, 17.2 ± 1.9 pmol/min/mg in A549), while
34
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
HeLa, MCF-7, and L929 exhibited increased AS activities (5.5 ±
0.2 pmol/min/mg in HeLa, 229.3 pmol/min/mg in MCF-7, and
258.9 pmol/min/mg in L929).
□ Control
E 2 cytokines 300
250
0 3
-§ 200
c
'E
o 150
E
_ Q .
S' 100
’ >
to 50
CO
<
^ 7 7 1 L / / / I I / / / _ _
HeLa MCF-7 L929
■ ■ (2.5ng/ml
T N F -a )
CHO MDCK A549
-50
Fig 12. AS activity in cells treated with Interferon-gamma (IFN-y)
50 unit/ml and Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml for
24 hours. Note L929 cells, TNF-a 2.5 ng/ml was used. The results
were compared with the control groups. MDCK, CHO, and A549,
showed similar AS activities; while HeLa, MCF-7 and L929 cells
exhibited the increase in AS activity.
3.2-4. Influence of rADI and Cytokines on AS Activity in
Different Cell Lines
The effects of rADI plus cytokines on AS activity were
investigated. Interferon-gamma (IFN-y) 50 unit/ml and Tumor
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Necrosis Factor-alpha (TNF-a) 5 ng/ml were used. Cell were
treated with rADI, 1 mU/ml, for 24 hours. Again, due to the
severe cytotoxicity of TNF-a 5 ng/ml in L929, a lower dose of
TNF-a 2.5 ng/ml was used.
Among these six cell lines, three rADI-sensitive cell lines
showed the lowest AS activity in this combination treatment
group compared to the other treatment groups (i.e., control group,
rADI group, and cytokine group) (-1.7 ± 0.1 pmol/min/mg in
MDCK, -12.1 ± 0.1 pmol/min/mg in CHO, -7.1 ± 0.1
pmol/min/mg in A549). While in the rADI-resistant cells, the
highest AS activity was exhibited in the combination treatment
group compared to other treatment groups (74.5 ± 1.8
pmol/min/mg in HeLa, 292.5 ± 15.1 pmol/min/mg in MCF-7,
351.2 ± 23.7 pmol/min/mg in L929 TNF-a 2.5 ng/ml group).
The data of L929 cells with the same dose treatment as
other cells (IFN-y 50 unit/ml, TNF-a 5 ng/ml, and rADI 1 mU/ml)
was also compared with the lower dose treatment (IFN-y 50
unit/ml, TNF-a 2.5 ng/ml, and rADI 1 mU/ml). L929 are resistant
to rADI treatment, the control and rADI had similar results (25.3
± 2.8 pmol/min/mg in control group of high dose treatment group,
8.9 ± 0.9 pmol/min/mg in ADI group). However, the AS activity
in cytokine treated and combination groups were much lower than
that in TNF-a 2.5 ng/ml group (59.1 ± 0.9 pmol/min/mg in
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
cytokines group in TNF-a 5 ng/ml treatment group and 90.9 ± 2.3
in combination group).
U )
£
" c
I
o
£
Q .
'>
o
ra
CO
<
400
350
300
250
200
150
100
50
0
-50 MDCK
□ Control
I2I ADI
H cytokines
B cytokines + ADI
L A J u r
CHO
i
JZ 3zzb= -
A549 HeLa MCF-7 L929
(2.5ng/ml
H M F-a)
Fig 13. AS activity in cells treated with Interferon-gamma (IFN-y)
50 unit/ml and Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml and
rADI 1 mU/ml each alone or together for 24 hours. In L929 cells,
however, TNF-a 2.5 ng/ml instead of 5 ng/ml was used. This is
because L929 is sensitive to TNF-a, high dose of TNF-a would
cause severe cytotoxicity to L929. Three rADI-sensitive cells
(MDCK, CHO, and A549) showed low AS activity in the
combination treatment groups, while in rADI-resistant cells
(HeLa, MCF-7, and L929), the highest AS activities were
exhibited the in the combination treatment groups compared to
other treatment groups.
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
O )
E
"c
E
o
E
Q .
O
CO
< * )
<
400
350
300
250
200
150
100
50
□ control
0 ADI
H cytokines
B cytokines + ADI
L929 (5ng/ml TNF- a) L929 (2.5ng/ml TNF- a )
Fig 14. AS activity in L929 cells treated with different dosages.
One set was treated with interferon-gamma (IFN-y) 50 unit/ml
and Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml and rADI 1
mU/ml each alone or together for 24 hours, and the other set was
treated with TNF-a 2.5 ng/ml instead of 5 ng/ml. This is because
L929 is sensitive to TNF-a, high dose of TNF-a would cause
severe cytotoxicity. The two sets of the treatment groups showed
a similar trend. However, the AS activities in cytokine treated and
combination groups by using TNF-a 5 ng/ml exhibited much
lower than that in TNF-a 2.5 ng/ml group.
3.3 NO Study
3.3-1. NO in different ceil lines
The NO production without any treatment in the cell
homogenates of the six cell lines is shown in Fig. 15. Among the
six cell lines, A549 and MCF-7 cells had comparatively high NO
production: 106.9 ± 7.0 pmol/mg, 89.2 ± 11.3 pmol/mg. The NO
production of 43.9 ± 1.7 pmol/mg was observed in MDCK, 55.5 ±
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
B3001$$833C
4.7 pmol/mg in CHO, 74.8 ± 5.3 pmol/mg in HeLa. L929 had the
lowest NO production (7.5 ± 0.8 pmol/mg) among all the cell
lines.
120
100
80
60
40
20
0
MDCK CHO A549 HeLa MCF-7 L929
Fig 15. Nitric oxide (NO) production in cells without any
treatment. NO was measured when the cells reached confluence at
day 3.
3.3-2. Influence of rADI on NO production
After 24 hour treatment with 1 mU/ml rADI, the cells
responded differently in NO production. Most of the cell lines,
MDCK, CHO, HeLa, and L929, showed decreased NO production
(14.5 ± 0.4, -397.7 ± -9.6, 3.1 ± 0.2, and -18.2 ± -0.6 pmol/mg)
(fig. 16). In CHO cells, the decrease of NO production is
considered as not detectable. A small increase in NO production
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
due to rADI was shown in A549 (131.3 ± 23.7 pmol/mg) and
MCF-7 (121.2 ± 16.0 pmol/mg).
□ control
0 A D I
150
0 5
------------ ' r~ \^ A
A549 HeLa MCF-7 L929 -50 -M D C K
Q.
-150
~ -250
-350
-450
Fig 16. NO production in cells treated with rADI 1 mU/ml for 24
hours. The results were compared with the control groups without
any treatment. MDCK, CHO, HeLa, and L929 showed decrease
NO production; while A549 and MCF-7 cells exhibited a small
increase in nitric oxide production. Notably it was a huge increase
of AS activity in HeLa cells. Huge decrease of NO production in
CHO cells would be considered as not detectable.
3.3-3. Influence of cytokines on NO production
The effects of cytokines on NO production were also
investigated. Still, Interferon-gamma (IFN-y) 50 unit/ml and
Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml were used. The
40
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lower dose of TNF-a 2.5 ng/ml instead of 5 ng/ml was used in
L929 cells.
MDCK, CHO, and HeLa did not show any induced NO
production (41.1 ± 0 .5 pmol/mg, -31.0 ± -1.5 pmol/mg, 72.5 ± 2.4
pmol/mg). A549 cells showed a small amount of induction in NO
(154.0 ± 4.7 pmol/mg), and MCF-7 cells exhibited an increase
compared to control (180.1 ± 6.4 pmol/mg). Among the six cell
lines, L929 had the highest, over 100 fold increase of NO
production by cytokine stimulation (1221.8 ± 196.7 pmol/mg).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
□ control
SI cytokines
1500
1300
T O
1100
900
700
500
Q .
300
100
-100 MDCK ~ CHO A549 HeLa MCF-7 L929
(2.5ng/ml
T N F -a )
Fig 17. Nitric oxide production in cells treated with
Interferon-gamma (IFN-y) 50 unit/ml and Tumor Necrosis
Factor-alpha (TNF-a) 5 ng/ml for 24 hours. There is one
exception in L929 cells, TNF-a 2.5 ng/ml instead of 5 ng/ml was
used. The results were compared with the control groups without
any treatment. MDCK, CHO, and HeLa did not show any induced
NO production. A549 had small induction of NO, and MCF-7
showed about twice amount of NO production by cytokines. L929,
however, exhibited dramatically increase in NO production.
3.3-4. Influence of rADI and cytokines on NO production
The effect of rADI plus cytokines on NO production was
determined. Again, Interferon-gamma (IFN-y) 50 unit/ml and
Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml were used. Cells
were treated with 1 mU rADI /ml for 24 hours. In L929 cells,
42
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TNF-a 2.5 ng/ml instead of 5 ng/ml was used in order to avoid the
severe cytotoxicity.
In five out of the six cell lines (MDCK was the exception),
rADI lowered the NO production induced by cytokines (-273.6 ±
-17.9 pmol/mg in CHO, 71.0 ± 4.9 pmol/mg in A549, 9.8 ± 0.5
pmol/mg in HeLa, 122.6 ± 6.6 pmol/mg in MCF-7, 44.8 ± 2.5
pmol/mg in L929). Some of them, such as CHO, A549, and HeLa,
even decreased to a lower level than that in the respective control
group. The only increase in NO production with combination
treatment of rADI and cytokines was in the MDCK cell line
(114.3 ± 3.8 pmol/mg).
Once more, the data of L929 cells with the same dose
treatment as other cells (IFN-y 50 unit/ml, TNF-a 5 ng/ml, and
rADI 1 mU/ml) was also compared with that of a lower dose
treatment (IFN-y 50 unit/ml, TNF-a 2.5 ng/ml, and rADI 1
mU/ml). L929 cells with severe cytotoxic exposure had extremely
high NO production (8224.7 ± 1339.8 pmol/mg) compared to that
in the control group (9.2 ± 0.3 pmol/mg). rADI alone, however,
did not affect the NO production (9.0 ± 0.2 pmol/mg). rADI
inhibited the induced NO production by cytokine treatment
(1661.4 ± 144.2 pmol/mg).
43
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□ control
0 A D I
H cytokines
B cytokines + A D I
1350
1150
0 3
950
750
Q .
550
350
150
-50
A549 HeLa MCF-7 L929
.............. - ■ - • - (2.5ng/ml
T N F-a)
-250
-450
Fig 18. NO production in cells treated with Interferon-gamma
(IFN-y) 50 unit/ml and Tumor Necrosis Factor-alpha (TNF-a) 5
ng/ml and rADI 1 mU/ml each alone or together for 24 hours. In
L929 cells, however, TNF-a 2.5 ng/ml instead o f 5 ng/ml was
used. This is because L929 is sensitive to TNF-a, high dose of
TNF-a would cause severe cytotoxicity to L929. Five out of the
six cell lines (MDCK was the exception), rADI exhibited the
strong ability to lower NO production induced by cytokines.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
□ control
0 ADI
E S cytokines
B cytokines + A D I
8900
O )
7900
§_ 6900
5900
■o 4900
o
3900
2900
1900
900
-100
L929(5ng/ml T N F -a ) L929 (2.5ng/ml T N F -a )
Fig 19. NO production in L929 cells treated with different
dosages. One set was treated with interferon-gamma (IFN-y) 50
unit/ml and Tumor Necrosis Factor-alpha (TNF-a) 5 ng/ml and
rADI 1 mU/ml each alone or together for 24 hours, and the other
set was treated with TNF-a 2.5 ng/ml instead of 5 ng/ml. This is
because L929 is sensitive to TNF-a, high dose o f TNF-a would
cause severe cytotoxicity. The two sets of the treatment groups
showed the similar trend. However, the NO production in
cytokine treated and combination groups by using TNF-a 5 ng/ml
exhibited much higher than that in TNF-a 2.5 ng/ml group.
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4. Discussion
4.1 Cells resistant to rADI have high A S activity
It appears that a correlation exists between AS activity and
rADI-resistance [Shen et al., 2003] in the six cell lines that were
examined (Fig. 9 and 11). Among the rADI-resistant cells (HeLa,
MCF-7, and L929), high induction of AS activity by rADI was
observed in HeLa. This might be because when arginine, the
amino acid that cells need to survive, was depleted by rADI, the
cells then needed to regenerate more arginine in order to stay
alive. The regeneration of arginine occurs via AS activity in the
arginine-citrulline cycle. The higher the AS activity, the more
arginine can be regenerated. This could be the reason why HeLa
cells with rADI-induced AS activity are resistant to rADI. MDCK,
CHO, and A549 showed the lowest AS activity in the control
group (without treatment), and AS activity was further reduced
upon treatment with rADI (Fig. 11). Consequently, arginine could
not be regenerated in these three cell lines and cell death occurred
with rADI treatment. Although AS activity in MCF-7 and L929
was also reduced upon rADI treatment, their AS activity was
sufficient enough to regenerate a significant amount of arginine to
keep them alive during rADI treatment.
46
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4.2 Induction o f argininosuccinate synthetase activity by
cytokines.
The data here demonstrated that cytokines could
effectively increase AS activity in HeLa, MCF-7, and L929 cells
(Fig. 12). Similar results were also seen in vascular smooth muscle
cells [Nussler et al., 1994] [Hattori etal., 1994], AS enzyme activity
and AS mRNA are markedly induced in cells when treated with
cytokines, while AL, another urea cycle enzyme responsible for
the regeneration of arginine from citrulline, is constitutive during
cytokine treatment [Nussler et al., 1994] [Hattori et al., 1994]. This
might be because arginine, the substrate for NOS, is consumed
when cytokines induce iNOS activity (Fig.3); arginine
regeneration activity is therefore required. Thus, AS activity,
which is responsible for arginine regeneration, is enhanced.
However, there was no obvious increase in AS activity induced by
the cytokines in MDCK, CHO, and A549 cells (Fig. 12). It is
possible that either iNOS induction by cytokines (Fig. 17) or
regeneration of arginine is inefficient.
4.3 rADI abolishes nitric oxide production by iNOS
From Figure 17, rADI could even lower the baseline NO
production under the conditions without any NO induction in
almost all the six cell lines, especially in MDCK, CHO, HeLa,
47
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and L929 cells. However, an obvious increase of NO production
induced by cytokine treatment, 50 U/ml IFN-y and 5 ng/ml TNF-a
(2.5 ng/ml TNF-a in L929 cells) was only seen in A549, MCF-7,
and L929 cells (Fig. 17). rADI, on the other hand, could
effectively reduce NO production induced by cytokines in the
CHO, A549, HeLa, MCF-7, and L929 cell lines (Fig. 18). This
might be because when rADI depleted arginine, the substrate of
NO synthase, nitric oxide (NO) could not be synthesized due to
the lack of this amino acid (Fig.3). It has been shown that an
intraperitoneal injection of rADI significantly suppressed the rise
of blood nitrite/nitrate levels that were induced by the systemic
administration of bacterial endotoxin LPS to mice, resulting in an
improvement of the survival rate [Noh et al., 2002]. The results
suggest that the depletion of blood arginine with an
arginine-metabolizing enzyme, such as ADI, could suppress
excessive production of NO that is caused by inducible NOS
(iNOS) during the endotoxemia. The only exception of this NO
suppression effects by rADI among these tested six cell lines was
seen in MDCK cells. However, rADI still has great potential to be
used as a new approach to control NO-related diseases, such as
sepsis.
48
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4.4 Correlation between rADI, cellular argininosuccinate
synthetase (AS) activity and nitric oxide (NO)
Taken the whole results together from the combination
group (rADI plus cytokines group), rADI could effectively inhibit
the NO production in most of the cell lines, including CHO, A549,
HeLa, MCF-7, and L929 cell lines. On the other hand, AS activity
was low in the rADI-sensitive cell lines, such as CHO, and A549.
However, for rADI-resistant cell lines (HeLa, MCF-7, and L929),
though the NO production could be inhibited by rADI, the AS
activity was even higher in the combination treatment group of
cytokines plus rADI. The regulation of NO production via iNOS
by rADI seems not to have a direct correlation with AS activity in
most, if not all, cells.
Furthermore, the induction of AS is not blocked by
NG-monomethyl-L-arginine, a potent inhibitor of NOS, indicating
that AS induction is not the consequence of depleting cellular
arginine levels by the reaction of NOS [Nussler et al., 1994].
Plasma levels of arginine are critical for NO synthesis, therefore,
enhanced cellular capacity to regenerate arginine from citrulline
could play a significant role in regulating NO production,
especially under conditions where the inducible isoform of NOS
is expressed [Flodstrom et al., 1995].
49
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4.5 Cytoprotective effect o f arginine deiminase (rADI)
Several reports have demonstrated the cytoprotective
effect of ADI in vitro and in vivo. In human prostate cancer cells,
ADI-induced arginine depletion may inhibit protein synthesis, and
result in the protection of taxol-induced apoptosis that requires
new protein synthesis [Kang et al., 20001. ADI also protects mice
from the lethal effects of TN F-a and endotoxin [Thomas et al.,
2002].
Also, in our results, L929 cell in the combination group
(cytokines plus rADI) was 1.5 times higher than that in the
cytokine treated group, where the toxicity is crucial to
TNF-a-sensitive L929 cells Table. 1). It seems that rADI could
also protect L929 cells from death due to the cytokine-induced
cytotoxicity apoptosis [Humphreys and Wilson, 1999]. Interestingly,
another finding is that the L929 cell number in the rADI treated
group was always a bit more than that in the control group
(Table. 1). However, the cause of the increase in cell number in
rADI-treated L929 cells is still not known.
4.6 Cytotoxicity caused by Tumor Necrosis Factor-alpha in
L929
Comparing all the data between a high dose treatment of
TNF-a (5ng/ml) and a low dose treatment of TNF-a (2.5ng/ml) in
50
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L929, a similar trend in AS activity and NO production can be
observed. However, almost four times more cells survived in the
lower dose treatment than in the high dose treatment (Table. 1).
The cells did not look healthy at all in the high dose treatment
group. The NO production was extremely high in the groups that
were treated with a high cytokine dose. This result might be
because more cells were under going apoptosis due to the
cytotoxicity, and hence produced more NO. Thus the L929 data
from the lower dose treatment (TNF-a 2.5ng/ml) was preferred
for the comparison with other cell lines.
4.7 Further consideration
4.7-1. Selectivity of ADI
Is ADI a selective NOS inhibitor or not? Selective iNOS
inhibition might prove to have clinical applications as it prevents
the decrease in GFR following LPS, even after renal failure is
established. Treatment with a non-selective NOS inhibitor in
septic patients should be reconsidered [Schwartz et al., 2001].
Therefore, if ADI could act as a iNOS inhibitor, including the
selective inhibition of iNOS while preserving eNOS activity
[Shao et al., 2001; Strunk et al., 2001; Wu et al., 1996], rADI would
have more potential to become a effective therapeutic drug.
51
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Further study of ADI must be explored in order to apply rADI
properly in clinical use.
4.7-2. Gene expression and protein regulation
The relationship among iNOS, AS mRNA, and protein
expression by cytokine treatment has been previously
demonstrated. For example, when neuronal PC 12 cells that had
been differentiated with nerve growth factor were treated with
interferon-y (IFNy) and tumor necrosis factor-a (TNFa), iNOS and
AS mRNAs and proteins were markedly induced, with AL mRNA
and protein being weakly induced [Zhang et al., 2000] [Braissant et
al., 1999] [Flodstrom et al., 1995]. However, the information
regarding the different regulation pathway, that of ADI-mediated
gene-regulation, is still not clear. Understanding the expression
and regulation of rADI effects on cells could help to develop ADI
as a powerful therapeutic drug.
4.7-3. Localization of AS, NOS enzymes, and arginine
transporters
Several studies have been reported on the localization of
AS, NOS, and arginine transporters that correlate with the
physiological activities. The colocalization of argininosuccinate
synthetase with nitric oxide synthase in caveolae in some brain
52
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regions has been demonstrated [Arnt-Ramos et al., 1992], [Flam et
al., 2001], [Isayama et al., 1997]. However, many neurons were
found to contain one of these two enzymes, but not the other.
Thus some nitric oxide synthase-containing neurons might be able
to recycle citrulline via argininosuccinate, while others can not.
Furthermore, in endothelial cells, the intracellular arginine is
sequestered in one or more pools that are poorly accessible to
eNOS, whereas extracellular arginine transported into the cell is
preferentially delivered to eNOS. Under this model, a plasma
membrane arginine transporter must be in close spatial alignment
with or directly linked to the eNOS protein [McDonald et al., 1997].
To identify the physiological importance of NO and
argininosuccinate in several regions is helpful to understand more
about the mechanism of rADI effects on cells.
4.8 Conclusion
There seems to be a correlation between AS activity and
rADI-resistance in the six cell lines that were tested. Cells with
high or inducible AS activity, such as HeLa, MCF-7, and L929,
possess a high ability to regenerate arginine, and therefore these
cells are resistant to rADI treatment. Induced AS activity by
cytokines was observed in several sutdies. rADI could inhibit the
NO production via iNOS, and its cytoprotective effects were also
53
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seen. However, rADI effects on AS activity does not follow the
same trend as the inhibition on NO production. Thus, it would be
interesting to understand how will the ADI treatment affect this
pathophysiological process. rADI does have a lot of potential to be
developed into a novel therapeutic drug for the treatment of cancer
and other diseases caused by the over-production of nitric oxide. It
would also be beneficial if the mechanism underlying the effects
caused by rADI in cells is fully understood to further develop
rADI for therapeutic use.
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Asset Metadata

Creator Lin, Wen-Chun (author)

Core Title Arginine deiminase-mediated modulation of argininosuccinate and nitric oxide synthesis in cultured cell lines

School Graduate School

Degree Master of Science

Degree Program Pharmaceutical Sciences

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

Tag Health Sciences, Pharmacology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Shen, Wei-Chiang (committee chair), Ann, David (committee member), Okamoto, Curtis T. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-305553

Unique identifier UC11337045

Identifier 1416565.pdf (filename),usctheses-c16-305553 (legacy record id)

Legacy Identifier 1416565.pdf

Dmrecord 305553

Document Type Thesis

Rights Lin, Wen-Chun

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