Savaraj et al.indd

Drug Target Insights 2007: 2 119–128 119

REVIEW

The Relationship of Arginine Deprivation, Argininosuccinate
Synthetase and Cell Death in Melanoma
Niramol Savaraj1, Chunjing Wu2, Marcus Tien Kuo3, Min You2, Medhi Wangpaichitr2,
Carlos Robles1, Seth Spector1 and Lynn Feun2
1VA Medical Center, Hematology-Oncology, Miami, Florida, U.S.A.
2University of Miami, Hematology-Oncology, Miami, Florida, U.S.A.
3M.D. Anderson Cancer Center, Molecular Pathology, Houston, Texas, U.S.A.

Abstract: It has been shown that melanoma cells do not express argininosuccinate synthetase (ASS) and therefore are unable
to synthesize arginine from citrulline. Depleting arginine using pegylated arginine deiminase (ADI-PEG20) results in cell
death in melanoma but not normal cells. This concept was translated into clinical trial and responses were seen. However,
induction of ASS expression does occur which results in resistance to ADI -PEG20. We have used 4 melanoma cell lines to
study factors which may govern ASS expression. Although these 4 melanoma cell lines do not express ASS protein or mRNA
as detected by both immunoblot and northernblot analysis, ASS protein can be induced after these cells are grown in the
presence of ADI-PEG20, but again repressed after replenishing arginine in the media. The levels of induction are different
and one cell line could not be induced. Interestingly, a melanoma cell line with the highest level of induction could also be
made resistant to ADI-PEG20. This resistant line possesses high levels of ASS mRNA and protein expression which cannot
be repressed with arginine. Our study indicates that ASS expression in melanoma cells is complex and governed by bio-
chemical parameters which are different among melanoma cells.

Keywords: Melanoma, arginine deiminase, argininosuccinate synthetase.

Introduction
Treatment of advanced malignant melanoma has not signifi cantly improved in the past 20 years. Most
chemotherapeutic agents have response rates of 15–20% or less (DeVita et al. 2001). Immunotherapeutic
agents including interleukin-2 , adoptive immunotherapy, interferon alpha and vaccine therapy may
produce signifi cant responses in a very small subset of patients, but their overall response rate is not
better than chemotherapy (Brinckerhoff et al. 2000; Riker et al. 2007). Combinations of chemotherapy
and immunotherapy also have not produced signifi cant response rates in randomized trials (Keilholz
and Gore, 2002; Punt et al. 2006; Sasse et al. 2007). Recently, blocking of Raf signaling has been
explored for the treatment of melanoma (Alsina et al. 2003; Hoefl ich et al. 2006; Scott et al. 2000;
Sridhar et al. 2005), however, the fi rst generation of Raf inhibitors such as Sorafenib has not shown
signifi cant antitumor activity in malignant melanoma. Thus, new effective agents are needed for the
treatment of this disease. We and others have shown that melanoma cell lines do not express arginino-
succinate synthetase (ASS) (Ensor et al. 2002; Feun and Savaraj, 2006; Scott et al. 2000; Shen et al.
2006; Wheatley, 2005; Wheatley and Campbell, 2002; Wheatley and Campbell, 2003) a key enzyme
which converts citrulline to argininosuccinate. Argininosuccinate is then converted to arginine by
argininosuccinate lyase. Thus, melanoma cells are particularly vulnerable to arginine depletion while
normal cells are able to survive. In fact, exposure of melanoma cells to arginine deiminase(ADI), an
enzyme which catalyzes the hydrolysis of arginine to citrulline results in cell death (Ensor et al. 2002;
Shen and Shen, 2006; Sugimura et al. 1992; Takaku et al. 1995; Takaku et al. 1993). This laboratory
fi nding has been developed into clinical trial, and antitumor activity has been documented in melanoma
patients (Ascierto et al. 2005; Feun and Savaraj, 2006). However, not all patients responded to treatment
and the duration of response varied. In this communication we explore the role of ASS gene expression
in ADI response/resistance using 4 melanoma cell lines as models.

Correspondence: Nirmal Savaraj, VA Medical Center, Hematology-Oncology, 1201 NW 16th Street, Miami,
Florida 33125. Tel: 305-575-3143; Email: nsavaraj@med.miami.edu
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm

120

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Drug Target Insights 2007: 2

Material and Methods

Cell lines
Four human melanoma cell lines were used: A375,
SK-MEL-2, A2058 and MEL-1220. The fi rst three
cell lines (A375, Sk-Mel-2 and A2058) were from
ATCC, and MEL-1220 was established in our
laboratory from a subcutaneous biopsy of a
melanoma patient. This cell line produces melanin
pigment and is positive for S-100. A telomerase
immortalized normal foreskin fi broblast cell line
(BJ-1) was purchased from Clonetech to be used
as normal control. NSCLCS was derived from
metastatic adenocarcinoma to the brain from
primary lung cancer. This cell line grows in
monlayer and was positive for keratin and epithe-
lial membrane antigen. All cells were cultured in
MEM with 10% FCS and Penicillin/Streptomycin.
Cells were checked for mycoplasma every month
using MycoAlert Mycoplasma Detection Kit
purchased from Cambrex.

Reagent
ADI-PEG20 was supplied by Polaris Inc. L-
Arg-free Medium was purchased from Invitrogen.
ASS antibody was purchased from BD Bioscience.
ASS antibody was purchased from BD-Biosci-
ences. PARP antibody was purchased from Cell
Signaling and actin antibody was purchased from
Sigma.

Growth inhibitory effect
0.6 × 105 cells were seeded onto 24-well plates and
allowed 6 hr. for attachment. Various concentra-
tions of ADI were added to each well. Each
concentration was performed in duplicate. After
72 hr. exposure, cells were counted in the presence
of 0.2% trypan blue. The growth inhibitory effect
(ID50) was determined by plotting the number of
viable cells as a percentage of control against the
compound concentration.

Detection of apoptosis
Cells (1 × 104) were seeded onto Lab-Tek chamber
slides (NALGE NUNC International) and then
exposed to ADI-PEG20 at 0.1 ug/ml for 72 hrs. The
apoptotic cells were detected by an in situ end-
labeling assay using a kit from ONCOR. Briefl y,
cells were fi xed in 4% neutral buffered formalin,
treated with 2% hydrogen peroxide, and incubated

with terminal deoxynucleotide transferase enzyme
and digoxigenin-11-dUTP under a plastic coverslip
for 1 hr. Anti-digoxigenin peroxidase was applied
to the slide, followed by the chromogenic substrate
diaminobenzidine and counterstained with hema-
toxylin. Cells that underwent apoptosis showed dark
brown staining in the nuclei. We have also used poly
ADP-Ribose Polymerase cleavage assay to detect
apoptosis. During apoptosis ICE family such as
caspase-3 and caspase-7 cleave PARP to yield 85
and 25 kDa fragments. Briefl y, cells were treated
with ADI-PEG20 for 72 hrs, nuclear protein was
obtained and immunoblot was performed using
PARP rabbit polyclonal antibody purchased from
Cell Signaling.

Analysis of arginine and citrulline by
HPLC
Arginine and citrulline analysis was performed by
cation exchange Hewlett Packard 1100 Series
HPLC with a post-column derivitization(11). This
instrument utilizes a cation exchange column and
a guard column. The temperature was set at 34 °C
and the reactor temperature was set at 39 °C.
Amino acid standards arginine and citrulline were
prepared in Li220 diluent (all mobile phase
reagents were obtained as pre-mixed preparations
from Pickering Laboratories). The mobile phase
reagents were Pump A Li280, Pump B Li750,
Pump C RG003. The initial conditions were 100%
A for 12 min., followed by a linear gradient of 0-
15% B over the next 16 min. The mobile phase
was then switched to 92% B and 8% C and an
isocratic gradient was run for an additional 37 min.
A constant fl ow rate of 0.3 mL/minute was used.

ASS expression detected by reverse
transcriptase polymerase
chain reaction
Total RNA was extracted using the kit from Invit-
rogen. First-strand cDNA was generated from 0.2ug
of total RNA using MLV Reverse-transcription. The
amplifi cation was carried out using the following
primers: forward primer: GGCCAAAAAGGT-
GTTCATTG (nt: 240–259); reverse primer: ATTC-
CAATGAAGCGGTTCTC (nt: 883–902) and set
as follows: denature: 45 sec. at 94 °C, annealing
45 second at 60 °C and extension 1 min. at 72 °C
for a total of 30 cycles. GAPDH was used as
control. The primers for GAPDH were as follows:

121

Argininosuccinate Synthetase in Melanoma

Drug Target Insights 2007: 2

forward primer: GAAGGTGAAGGTCGGAGTC;
reverse primer: CAAAGTTGTCATGGAT-
GACC.

Southernblot analysis of ASS gene
10 ug of nuclear DNA were digested with EcoRI
restriction enzyme and electrophoresed using 0.8%
agarose gels at 3 cm/V for 10–16 hrs in TAE buffer
(40 mM Tris-acetate, 1 mM EDTA, pH8.0). The
nuclear DNA was transferred onto a nylon fi lter
(Hybond-N; Amersham) and hybridized with
32P-dCTP ASS probe overnight, washed, and auto-
radiography was performed. ASS probe was gener-
ated from BJ-1 cell line. Briefl y, the ASS PCR
product was obtained using the primers mentioned
above, then eluted from the agorose gel using the
kit from Qiagen and labeled by 32P-dCTP using
random oligolabeling technique.

Northernblot analysis of ASS gene
Total RNA was extracted using the kit from
Invitrogen. 10 ug total RNA was separated in 1%
agarose gel in MOPS -formaldehyde buffer, trans-
ferred to a nylon membrane (Hybond-N; Amer-
sham) and hybridized with 32P-dCTP labeled ASS
probe. Autoradiography was carried out.

Westernblot analysis of ASS
Cells were lysed with RIPA buffer (10 mMTris
pH7.4 100 mM NaCl, 1 mM EDTA, 20 mM
Na4P2O7, 2 mM Na3VO4, 1% NP-40, 0.5% deoxy-
cholate, 1 mM PMSF) and protease inhibitor
cocktail from Sigma, passed several times through
a 23G needle, and centrifuged. The total protein
was separated on 12% SDS-PAGE, transferred
onto membrane and immunoblot with a specifi c
antibody and detected by chemiluminescence.

Construction of human argininosucci-
nate synthetase expression vector
To construct pcDNA3-ASS, we amplified the
human argininosuccinate synthetase mRNA
sequence from 79 to 1314 nt (using GenBank
accession number X01630 as the reference) by
PCR with primer pairs containing sequences
5’GCGGCCGC TCCAGCAAAGGC TCCGTGG
(sense; underscore sequence contains the Not I site)
and 5’-GCGGCCGCTATTTGGCAGTGACCTTGC
(antisense; underscore sequence contains Not I

site) using human pancrease cDNA (Clonetech,
Mountain View, CA) as the template. The PCR
product was cloned into PCR®II-TOPO vector
(Invitrogen). The NotI fragment containing the
respective cDNA was then transferred into the NotI
site of CIN-HA-pcDNA3 vector, which contains
a hemagglutinin tag, enhancer CIN sequence for
expression, and a neomycin resistance marker for
transfection selection.

Transfection of ASS gene. ASS cDNA
was introduced into A375 and A2058 cell line
using lipofectamine from Invitrogen and selected
with G-418. These transfectants were then tested
for their sensitivity to ADI-PEG20.

Results

Growth inhibitory effect of ADI-PEG20
The growth inhibitory effect as well as the
arginine and citrulline concentrations in the media
are shown in Table I. The ID50 ranged from 0.05–
0.08 ug/ml. However, there are no viable cells in
all three cell lines (A375, Sk-Mel-2 and Mel-1220)
after exposure to ADI-PEG20 for seven days,
whereas in A2058 there is 2–3% viable cell left.
From the ID50 results, it appears that in Sk-Mel-2
and A2058 cell lines require more ADI-PEG20 to
deplete arginine in the media. At 72 hr. there is still
arginine remaining in the media (12.8 uM for Sk-
Mel-2 and 23.5 uM for A2058) whereas in A375
and MEL-1220 there is no detectable arginine level
in the media. These fi ndings may be related to the
capability of the intracellular machinery to maintain

ASS

ACTIN

A
37

S
k-

M
el

-2

A
20

M
E

L-
12

B
J-

N
S

C
LC

Figure 1. Immunoblot of ASS protein using commercially available
monoclonal antibody (BD-Bioscience). All four melanoma cell lines
(A375, Sk-Mel2, A2058 and MEL-1220) do not express ASS protein
whereas NSCLCS and BJ-1 possess high levels of ASS protein.
Actin was used as control.

122

Savaraj et al

Drug Target Insights 2007: 2

arginine either by degradation of certain unessential
proteins or turning on ASS or other unknown
mechanisms. All these 4 melanoma cell lines do
not express ASS protein (as detected by western-
blot, Fig.1) and neither at the transcriptional levels
as detected by northernblot analysis. This data also
correspond to what has been reported in the litera-
ture (Dillon et al. 2004; Ensor et al. 2002). However,
the ASS DNA can be detected readily by southern-
blot analysis (Fig. 2). Thus, lack of ASS expression
is not from gene deletion. In contrast, degradation
of arginine by ADI-PEG20 did not have growth
inhibitory effect on normal fi broblast BJ-1 and
NSCLCS cell line (Table 1). These two cell lines
possess high levels of ASS and hence are able to
synthesize arginine from citrulline. These two cell
lines also have arginine in the media at 0.05 ug/ml
of ADI-PEG20. At this concentration there is no
arginine in the media when EMEM media was
incubated with ADI-PEG20 alone with no cells for
3 days.

ADI induces apoptosis in melanoma
cell line
We have exposed melanoma cells to arginine free
EMEM media with citrulline and NH4Cl supple-
ment or ADI at 0.05 ug/ml for 3 days and assay for
apoptosis. The results are shown in Figure 3A & 3B.
All melanoma cell lines undergo apoptosis by both
PARP assay and in situ end labeling assay whereas
BJ-1 and NSCLCS cells do not undergo apoptosis
(data not shown).

Arginine deprivation induces mela-
noma ASS gene expression
It has been shown that arginine levels can
regulate ASS expression in lymphoblastoma cells

and epithelial cells (Jackson et al. 1996; Philip
et al. 2003). However, whether this phenomenon
occurs in melanoma cells which lack ASS expres-
sion is not known. We have studied this possibility
by exposing four melanoma cell lines (A375,
Sk-Mel-2, A2058 and MEL-1220) to ADI-PEG20
for 3 days and assay for ASS expression . The
results are shown in Figure 4. It is of interest that
despite the similar levels of arginine deprivation,
the levels of ASS expression as detected by west-
ernblot are different. A2058 has more ASS expres-
sion (2.76 fold) followed by Sk-Mel-2 (2.03) and
A375(1.52) while MEL-1220 is unable to turn on
ASS gene. To further confi rm this fi nding we have
cultured all four cell lines in the arginine free
media with or without citrulline (0.4 uM) and
NH4Cl (0.4 uM) for 72 hr. as well as using MEM
media which has been incubated with 0.05 ug of
ADI-PEG20 for 72 hr. to deplete arginine. At 24,
48 and 72 hr., cells were harvested and assayed
for ASS expression. After 72 hrs cells were placed
on normal EMEM media. At 24, 48 and 72 hrs
after being in normal media cells were harvested

Table I. Growth Inhibitory Effect of ADI-PEG20.

Cell lines ID50(ug/ml) Arginine uM Citrulline uM
(72 h) (72 h)
A2058 0.088 ± 0.008 23.5 367.5
A375 0.055 ± 0.001 ND 317.5
Sk-Mel-2 0.07 ± 0.008 12.8 329.2
MEL-1220 0.09 ± 0.005 0 3
BJ-1 >1 1.6 404
NSCLCS* >1 20.27 603
*RPMI media (base line Arginine: 971uM)
EMEM media (base line Arginine: 400uM)

A
37

S
k-

M
el

-2

A
20

M
E

L-
12

20 B
J-

ASS

ACTIN

Figure 2. Southernblot Analysis of ASS in 4 melanoma cell lines and
BJ-1 cell lines. All fi ve cell lines show similar levels of ASS DNA.

123

Argininosuccinate Synthetase in Melanoma

Drug Target Insights 2007: 2

and assayed for ASS expression. The results are
shown in Figure 5. The three type of media yield
similar results. Similar to data in Figure 4, A2058
appears to have highest ASS levels in arginine
free media followed by Sk-Mel-2 and A375, while
MEL 1220 has no effect (data not shown). Thus,
our results indicate that the levels of arginine in
the media regulate ASS protein levels, and that
the amount of citrulline did not have any effect.
Interestingly, ASS gene expression cannot be
detected by northernblot analysis (Fig. 8), but
may be slightly increased by RT-PCR (data not
shown). Thus, it appears that increased translation
of ASS proceeds transcription.

Arginine deprivation does not induce
ASS expression in NSCLC
We further investigated whether arginine depri-
vation also induced ASS expression in tumor
cell lines which constitutively express ASS. We
have chosen NSCLCS which was established

from metastatic adenocarcinoma of the lung to
the brain. This cell line expresses ASS and is not
sensitive to ADI-PEG20. Cells were seeded in
arginine free media with citrulline supplement
or ADI-PEG20 treated media to degrade all
arginine for 3 day and assay for ASS expression
by westernblot. The results are shown in
Figure 6. ASS expression is similar in normal
media or arginine depleted media. Thus, it
appears that arginine deprivation does not have
effect on ASS expression in tumor cell lines
which constitutively express high levels of
ASS.

ASS gene transfection results
in resistance to ADI
We have transfected ASS cDNA into A375, and
A2058, and then assay for growth inhibitory effect
of ADI-PEG20. We are able to obtain only 2 fold
increase in ASS expression as shown by RT-PCR
and westernblot (Fig. 7A & 7B). The ID50 of these

A B

Figure 3A. In situ end labeling apoptosis assay in A375 cell line. A: control. B: after exposure to 0.08 ug/ml for 72 hr. Treated cells undergo
apopotosis shown brown staining in the nuclei.

116kD
89kD

Actin

- + - + - + - +ADI
A375 Sk-Mel-2 A2058 MEL-1220

PARP

Figure 3B. Apoptosis as detected by PARP cleavage in 4 melanoma cell lines (A375, Sk-Mel-2, A2058 and MEL-1220). Untreated cell
showed uncleaved PARP at 116 kD whereas treated cells showed cleaved PARP seen at 89 kD.

124

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Drug Target Insights 2007: 2

Generation of ADI-PEG20 resistant
cell line
Since we were unable to generate a stable ASS
transfected cell line with high levels of ASS expres-
sion, we have exposed A2058 cell lines to
ADI-PEG20 at 0.05 ug/ml for 5 days with 2 days
off × 6 weeks. We were able to establish A2058R
with high levels of ASS expression by northernblot
(Fig. 8A) and by westernblot analysis (Fig. 8B).
This cell line is resistant to ADI-PEG20 with ID50
>1 ug/ml. Interestingly, the levels of ASS protein
also are not affected by arginine deprivation
(data not shown).

Discussion
It has been reported that melanoma cell lines as
well as tumor samples do not express ASS and
thus are auxotrophic for arginine. Arginine depri-
vation using arginine deiminase or arginase has

ASS

ACTIN

ASS

ACTIN

ASS

ACTIN

A375

Sk-Mel-2

A2058

1 2 3 4 5 6 7

Figure 5. Immunoblot of ASS protein in 3 melanoma cell lines (A375,
Sk-Mel-2, A2058) before and after exposure arginine free media
supplemented with citrulline and NH4Cl for 24, 48 and 72 hrs. After-
ward, cells were washed, and replenished with normal EMEM media
for 24, 48 and 72hrs. Lane 1: control. Lane 2: 24 hrs on arginine free
media Lane 3: 48hrs on arginine free media Lane 4: 72 hrs in arginine
free media Lane 5: Removal of arginine free media and changed to
normal EMEM media for 24 hrs. Lane 6: 48 hrs in normal media.
Lane 7: 72 hrs in normal media. Similar results were obtained with
arginine free media with no citrulline supplement and ADI-PEG20
treated media.

ASS

Actin

1 2 3

NSCLCS

Figure 6. Immunoblot of ASS in NSCLCS: lane 1: control (normal
media ). lane 2: ADI-PEG20 treated media for 72 hr. Lane 3 : After
exposure to arginine free media with citrulline and NH4Cl
supplement for 72 hrs.

transfectants were 0.1 ± 0.05 ug/ml for A375, and
0.2 ± 0.05 ug/ml for A2058, respectively. Although
we are unable to yield high levels of ASS expres-
sion in these transfectants, this modest increase in
ASS expression does affect slightly the ID50 of
ADI-PEG20 with 2 fold increase.

A375 Sk-Mel-2 A2058 MEL-1220

ASS

ACTIN

- + - + - + - +ADI

Ratio of ASS 1.52 2.03 2.76 1
Figure 4. Immunoblot of ASS protein in 4 melanoma cell lines (A375, Sk-Mel-2, A2058 and MEL-1220) before and after exposure to
ADI-PEG20 for 3 days. The ratio of ASS depicts the intensity of ASS expression by densitometer after exposure to ADI versus control back-
ground. A2058 exhibited the highest level of expression whereas MEL-1220 did not express ASS after exposure to ADI-PEG20.

125

Argininosuccinate Synthetase in Melanoma

Drug Target Insights 2007: 2

been shown by several investigators to have
antitumor activity in melanoma cell lines as well
as in other tumor cell lines which lack ASS in vitro
(Dillon et al. 2004; Ensor et al. 2002; Gong et al.
2000; Miyazaki et al. 1990; Noh et al. 2004; Shen
et al. 2006; Sugimura et al. 1992; Szlosarek et al.
2006; Takaku et al. 1995; Takaku et al. 1993; Yoon
et al. 2007). Correlation of antiproliferative effect
of ADI-PEG20 with endogenous ASS levels also
has been reported (Ensor et al. 2002; Shen et al.
2003). However, these enzymes have a short half
life and high antigenicity which makes them not
suitable for in vivo use. To overcome this problem,
a pegylated form of ADI (ADI-PEG20) was
developed by Polaris Inc. and has been shown to
increase the half life as well as decrease the
antigenicity (Izzo et al. 2004). This compound has
been shown to have antitumor activity in vivo
(Ensor et al. 2002). Clinical trials also look
promising with antitumor responses seen in a
number of melanoma patients (Ascierto et al.
2005; Feun and Savaraj, 2006). However, not all
patients respond to ADI-PEG20 and resistance to
this compound does occur after treatment. Our
limited clinical data suggest that ASS expression
occurs in patients who develop resistance to
ADI-PEG20 (Feun and Savaraj, 2006). Thus, it
appears that exposure to ADI-PEG20 which
results in continuous arginine depletion in the
serum results in ASS expression and hence drug
resistance. In this communication, we have shown
that exposure of melanoma cells to ADI-PEG20
or arginine free media can result in ASS protein
production which again become negligible when
cells were exposed to normal media. Interestingly,
the levels of ASS protein being induced while

cells are deprived with arginine vary among
the 4 melanoma cell lines. A2058 has the highest
of ASS protein followed by Sk-Mel-2 and A375.
One melanoma cell line (MEL -1220 ) cannot
produce ASS protein despite being cultured in the
arginine depleted media. Interestingly, A2058 can
be selected to become resistant to ADI-PEG20
rather rapidly in 6 weeks. The primary mechanism
of resistance involves increase in ASS protein
production followed by ASS transcription as
detected by northernblot analysis. The resistance
also is irreversible despite replacement with
normal EMEM media. On the other hand,
MEL-1220 cannot be made resistant to ADI-
PEG20. These fi ndings may mirror what we have
found in melanoma patients. This is the fi rst report
which demonstrated that extracellular arginine
does control ASS protein in certain melanoma
cells. The underlying mechanism(s) on how argi-
nine controls ASS translation/transcription in
melanoma are not known. It is possible that when
the arginine levels is low, the cells will attempt to
increase the translation of ASS in order to
synthesize arginine from citrulline. We are

M
ar

ke
r

A
37

5-
2

A
37

5-
3

A
37

5-
4

A
37

A
20

58
-5

A
20

58
-6

A
20

ASS

GAPDH

ASS

Actin

1 2 3 4
Figure 7B. Immunoblot of ASS in control and transfected cells.
Lane 1: A-375. Lane 2: A375-3. Lane 3: A2058. Lane 4: A2058-6
(A375-3 and A2058-6 transfected with ASS).

Figure 7A. RT-PCR of ASS in A375 and A2058 and their ASS transfected clones A375-2, A375-3, A375-4. and A2058-5,A2058-6, respec-
tively. A375-3, and A2058-6 possess high levels of expression and were selected to study the growth inhibitory effect of ADI-PEG20.

126

Savaraj et al

Drug Target Insights 2007: 2

currently investigating both transcription and
translation control of ASS in both normal and
arginine free media. Interestingly, the levels of
induction are different among the melanoma cell
lines tested. It appears that the cell line which has
high levels of induction is capable of becoming
resistant to ADI -EG20 in a short period of time
(within 6 weeks). On the other hand, the cell line
which has no induction of ASS (MEL-1220) is
not capable of turning on ASS gene transcription
and becoming resistant to ADI-PEG20. Whether
these fi ndings occur in patients’ tumor samples is
not known. However, we have observed a mela-
noma patient who was ASS(-) prior to treatment
and become ASS positive at the time of treatment
failure which suggest that arginine deprivation

can result in ASS expression. As mention earlier,
the mechanism by which arginine in the media
control ASS expression is not known, we are
currently investigating how ASS gene transcrip-
tion is regulated in the presence and absence of
arginine.

ASS cDNA has been cloned in 1981 (Su et al.
1981). The kinetic properties of ASS enzyme has
been extensively studied and the crystal structure
in bacteria has been identifi ed (Husson et al. 2003;
Ratner, 1973). The transcriptional and translational
control of ASS gene are not well understood and
appear to be tissue specifi c (Husson et al. 2003).
Several hormones such as glucocorticoids, gluca-
gons, insulin and fatty acid have been shown to
positively or negatively infl uence ASS expression
(Husson et al. 1975; Husson et al. 1986; Husson
and Vaillant, 1979; Husson and Vaillant, 1982; Lin
et al. 1982). Our preliminary data does not indicate
that these hormones infl uence ASS expression in
melanoma cells. However, arginine in the media
can mediate ASS expression in certain melanoma
cells. In contrast, arginine in the media has no effect
in A2058R (selected by ADI-PEG20) or NSCLCS,
both have high levels of ASS expression. This
fi nding is similar to the previous report (Boyce
et al. 1986) which showed that arginine mediated
ASS repression does not occur in canavanine
(arginine analog) resistant variant of RPMI 2650
cells which has high levels of ASS expression.

ASS

ACTIN
A

37
5

A
37

5+
A

D
I

S
k-

M
el

-2

S
k-

M
el

-2
+A

D
I

M
E

L-
12

M
E

L-
12

20
+A

D
I

A
20

58
+A

D
I

A
20

58
R

B
J-

1+
A

D
I

Figure 8A. Northernblot Analysis of ASS in a panel of melanoma cell line and BJ-1 cells. Lane 1: A375. Lane 2: A375 after exposure to
ADI-PEG20 for 72hrs. Lane 3: SK-Mel-2 Lane 4: Sk-Mel-2 after exposure to ADI-PEG20 for 72 hrs. Lane 5: MEL-1220. Lane 6: MEL-1220
after exposure to ADI-PEG20 for 72 hrs. Lane 7: A2058. Lane 8: A2058 after exposure to ADI-PEG20 for 72 hrs. Lane 9: A2058R. Lane 10:
BJ-1. Lane 11: BJ-1 after exposure to ADI-PEG20 for 72 hrs. Note: only A2058R and BJ-1 possess 1.9 kb ASS mRNA and there is no
differences in ASS mRNA in BJ-1 cells after exposure to ADI-PEG20.

1 2 3 4 5

ASS

ACTIN

Figure 8B. Immunoblot of ASS. Lane 1: A2058. Lane 2: A2058 in
arginine free media with out citrulline and NH4Cl2. Lane 3: A2058 in
arginine free media supplemented with citrulline and NH4Cl. Lane 4:
A2058R. Lane 5: BJ-1 cells. ASS protein in A2058R is only slightly
less than BJ-1.

127

Argininosuccinate Synthetase in Melanoma

Drug Target Insights 2007: 2

Nevertheless, the key question remains what are
the factors which govern ASS expression in mela-
noma cells. If these factors can be identifi ed, one
can attempt to repress the ASS expression and
hence evade ADI-PEG20 resistance.

It is not yet clear why transfection with ASS
cDNA in melanoma cells did not yield high levels
of ASS expression and we are unable to generate
stable transfected cell line(s). It is conceivable that
certain factors in melanoma cells prevent this
cDNA to express at high levels and/or arginine in
the media may also play a role in repressing the
expression of transfected ASS. We are currently
investigating these possibilities.

Acknowledgment
This work was supported by a grant (R01CA
109578) from NIH and a research grant from VA.
We would like to thank Polaris Inc. for providing
ADI-PEG20 and analysis of arginine and citrulline.

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/NOR <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>
/SVE <FEFF0041006e007600e4006e00640020006400650020006800e4007200200069006e0073007400e4006c006c006e0069006e006700610072006e00610020006e00e40072002000640075002000760069006c006c00200073006b0061007000610020005000440046002d0064006f006b0075006d0065006e00740020006d006500640020006800f6006700720065002000620069006c0064007500700070006c00f60073006e0069006e00670020006600f60072002000700072006500700072006500730073007500740073006b0072006900660074006500720020006100760020006800f600670020006b00760061006c0069007400650074002e0020005000440046002d0064006f006b0075006d0065006e00740065006e0020006b0061006e002000f600700070006e006100730020006d006500640020004100630072006f0062006100740020006f00630068002000520065006100640065007200200035002e003000200065006c006c00650072002000730065006e006100720065002e00200044006500730073006100200069006e0073007400e4006c006c006e0069006e0067006100720020006b007200e400760065007200200069006e006b006c00750064006500720069006e00670020006100760020007400650063006b0065006e0073006e006900740074002e>
/ENU <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>
>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice