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[CANCER RESEARCH 30, 768-772, March 1970]
Inhibitory Effect of L-Asparaginase in Lymphocyte
Transformation Induced by Phytohemagglutinin
Moriji Miura, Masami Mirano, Kumi Kakizawa, Akimitsu Merita, Tadaaki Uetani, and
Kazumasa Yamada
First Department of internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
SUMMARY
The effect of Escherichia coli L-asparaginase on phytohemagglutinin (PHA)-induced transformation of human
lymphocytes was studied. Transformation was remarkably
suppressed by the enzyme when it was added to the cul
ture not later than 24 hr after its initiation. The effective
concentration was more than 0.2 i.u./tube. Guinea pig
serum and L-0-aspartohydroxamic acid, an L-asparagine
analog, have a suppressive effect on lymphocyte transfor
mation in this system. PHA-treated lymphocytes became
sensitive to L-asparaginase 12 hr after the culture was
started, while no L-asparagine dependency was detected
at any stage of the culture. An addition of L-asparagine or
L-glutamine to the culture system treated with both PHA
and L-asparaginase at zero time caused the partial re
covery of thymidine-'H uptake.
Thus, it is concluded that one of the mechanisms in
hibiting PHA lymphocyte transformation by L-asparag
inase is due to the deprivation of exogenous L-asparagine
and i.-glutamine from the tissue culture medium.
INTRODUCTION
Many investigators have reported that L-asparaginase
inhibits tumor growth in the mouse (4-7, 15), rat (16), dog
(23), and human (13, 22), through the inhibition or dele
tion of tumor-specific metabolism relating to L-asparagine.
Becker and Broome (3) found, however, that L-asparag
inase affects nonneoplastic tissue such as regenerating
rat liver.
The biochemical similarities of PHA'-transformed lym
phocytes to regenerating rat liver subsequent to partial
hepatectomy regarding a state of the activated nucleic
acids (9, 12, 26) and protein synthesis (2, 14, 24, 25) lead
to a question of whether or not L-asparaginase can affect
the PHA-transformed lymphocytes. Astaldi et al. (1) re
ported that L-asparaginase could intensively inhibit biastic transformation of human peripheral lymphocytes in
the PHA culture system in vitro. McElwain et al. (17) re
ported that the lymphocytes from the L-asparaginasetreated patients showed no transformation by PHA. The
stimulation of lymphocytes by PHA to grow and divide in
' The abbreviations used are: PHA, phytohemagglutinin:
L-0-aspartohydroxamic acid.
Received June 2. 1969; accepted August 6, 1969.
768
LAHA,
vitro provides an adequate experimental system for the
study of growth control mechanism or nutritional require
ment of the cell.
This study was undertaken to investigate the influence
of L-asparaginase in the course of lymphocyte transforma
tion induced by PHA and, furthermore, to clarify the
mechanisms involved.
MATERIALS
AND METHODS
Leukocyte Preparation. Ten ml blood from healthy sub
jects with blood type O were obtained, with citric acid-trisodium citrate-dextrose solution as an anticoagulant. The
blood was mixed with 25% of its volume of 6% dextranphosphate-buffered saline (-) (Dextran: Meito Co. Ltd.,
Nagoya, Japan; M. W. 270,000, pyrogen free), and the
red cells were allowed to sediment at 37°for 40 to 50
min. The supernatant leukocyte-rich plasma was drawn
with a sterile pipet and subsequently left to stand at 37°
for 4 hr to eliminate as many neutrophils and monocytes
as possible. The supernatant was obtained which con
tained more than 50% lymphocytes. The lymphocyte-rich
plasma was then centrifuged for 10 min at 1200 rpm to re
move platelets. The cells remaining were suspended in
warm culture medium, and the concentration was ad
justed to 10 X 106/ml.
Culture Medium. Eagle's HeLa Medium (Grand Island
Biological Company, Grand Island, N. Y.) supplemented
with 10% inactivated fetal calf serum (Grand Island Bio
logical Company, Grand Island, N. Y.) was used.
PHA. One vial PHA-P (Difco Laboratories, Inc., De
troit, Mich.) was dissolved in 5 ml hemagglutination buf
fer (Difco). Before use, it was diluted 100-fold with Eagle's
HeLa medium, and 0.1 ml of this PHA solution was
added to each culture tube.
L-Asparagine and L-Glutamine. L-Asparagine (Daiichi
Pure Chemical Co., Ltd., Tokyo, Japan) and L-glutamine
(Kyowa Hakko Co., Ltd., Tokyo, Japan) were dissolved
with Earle's solution at concentrations of 5 and 30 mg/ml,
respectively. These were sterilized by passage through a
Millipore filter with a pore size of 0.45 ¿¿.
Guinea Pig Serum. Blood was aseptically obtained by
heart puncture, and then fresh serum was absorbed with
washed human blood cells at 4°for 30 min to eliminate
the natural heteroantibodies. This was repeated twice.
The absorbed sera were stored at —80°.
CANCER
RESEARCH
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L-Asparaginase and PHA-indiiced Lymphocyte Transformation
LAMA. LAHA (HONHCOCH2CHNH2COOH) (Tanabe Farm Co., Ltd., Osaka, Japan), one of the struc
tural analogs of L-asparagine (21) which possesses antineoplastic activity in certain rodent tumors (20), was sub
jected to the experiment on inhibition of PHA-induced
lymphocyte transformation. This compound was dissolved
with Earle's solution at a concentration of 10 mg/ml.
E. coli L-Asparaginase. One vial L-asparaginase (Kyowa
Hakko Co., Ltd., KW-020-1, 10,000 i.u./vial, Lot No.
1047, specific activity 184 i.u./mg protein) was dissolved
with 5 ml Earle's solution and kept at —80°
as a stock so
lution. Before use, it was diluted to a desired concentra
tion with Earle's solution.
Leukocyte Culture. One ml culture medium, 0.1 ml
leukocyte suspension, and 0.1 ml PHA-P solution were
incubated in a rubber-stoppered tube at 37°for 72 hr.
Procedures for L-AsparaginaseSensitivity Test and L-Asparagine Dependency Test. A triplicate of leukocyte cul
tures at various time intervals after the initiation served
as a set for these tests. To the first leukocyte culture tube
was added 0.1 ml Earle's solution; to the second tube 2.0
i.u. L-asparaginase in 0.1 ml Earle's solutions were added;
to the third tube 0.05 mg or 0.005 mg L-asparagine in 0.1
ml Earle's solution was added. Then, uridine-5-' H or thymidine-o-'H (Daiichi) was added to a final concentration
of 2 ^Ci/tube. At the end of the 12 hr incubation, the
cells were harvested, and the acid-insoluble residue was
obtained on a filter paper disc (Toyo Roshi Co., Ltd.,
Tokyo, Japan, No. 5 B) by successive treatment of the
cells with 5% cold trichloroacetic acid. The acid-insoluble
residue on the filter paper disc was dried. Each disc was
placed in a standard 20-ml low-potassium-counting vial,
and then 8 ml liquid scintillator A (PPO and POPOP in
toluene) were added. The samples were counted for 1 min
with a Model TEN, GSL-163 (Kobe Kogyo, Kobe, Japan)
liquid scintillation spectrometer.
A formula for the determination of L-asparaginase sen
sitivity is as follows.
cpm control culture
—cpm L-asparaginase-treated culture
X 100
cpm control culture
where in control culture, Earle's solution was added to the
culture instead of L-asparaginase. The values above 40%
represent L-asparaginase sensitivity and the values at 40%
or less represents L-asparaginase insensitivity.
A formula for the determination of L-asparagine de
pendency is as follows.
Blastic Transformation Rate. Blastic transformation rate
was calculated as follows. Smear preparations of the cul
tures were made after 72 hr incubation, and stained with
May-Griinwald-Giemsa solutions. Five hundred cells of
the lymphoid population per culture were then examined
and the percentage of blast-like cells was taken as the
blastic transformation rate.
RESULTS
Table 1 shows the effect of L-asparaginase on lympho
cyte transformation when incubated with the enzyme and
PHA for 72 hr. A remarkable suppression of lymphocyte
transformation was observed at concentrations more than
0.2 i.u./tube L-asparaginase. There was no inhibitory ef
fect of L-asparaginase at concentrations less than 0.02
i.u./tube. The lymphocytes left untransformed in the cul
tures were morphologically intact.
In the next experiment, inhibitory effect of L-aspara
ginase in relation to preincubation time with PHA was
studied as shown in Table 2. A remarkable suppression
was observed in cultures preincubated with PHA for less
than 24 hr.
In parallel to the above experiments, the effect of
guinea pig serum and LAHA, an L-asparagine analogy,
was studied (Table 3). Leukocytes were cultured with
guinea pig serum or LAHA in the presence of PHA for
Table 1
Inhibitory effect of L-asparaginase on human lymphocyte
transformation by PHA
(%°)L-Asparaginase
Experi
(i.u./tube)200i.u.0NTNTNT20I.U.00002.0i.u.00000.2i.u.NT*5.012.0
mentNo.910Test
control(%)83.583.095.093.0PHA
cellNo.1234PHA
' %, blastic transformation rate.
' NT, not tested.
Table 2
Inhibitory effect of L-asparaginase on human lymphocyte
transformation
by PHA: relationship
i.u./tube)Test
cpm L-asparagine-treated culture
—cpm control culture
X 100
cpm control culture
where in control culture, Earle's solution was added in
stead of L-asparagine. The values above 40% represent
L-asparagine dependency, and values at 40% or less rep
resent L-asparagine independency.
MARCH
with PHA
preincubation time
L-Asparaginase (20
cellNo.234PHAcontrol83.095.093.01201.4.Preincubationhr
with36
hr1.05 24
hr22.349.046.5PHA48
hr30.078.075.1
12.50
13.0time
1%, blastic transformation rate.
1970
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769
Miura, Mirano, Kakizawa, Marita, Uetani, and Yamada
72 hr, and blastic transformation rate was calculated. Both
materials inhibited the lymphocyte transformation; 0.1
ml the serum/tube or 0.1 mg LAHA/tube lowered the
blastic transformation rate to less than 50%.
L-Asparagine dependency and L-asparaginase sensitivity
were examined at various time intervals of culture of the
transforming lymphocytes (Table 4). Acquisition of Lasparaginase sensitivity was noted at 12 hr after leuko-
cytes were exposed to PHA. In contrast, L-asparagine
dependency was not observed at any stage of culture ex
amined.
Restorative effect of L-asparagine and L-glutamine on
the inhibition of PHA lymphocyte transformation by Lasparaginase was studied (Table 5). Leukocyte cultures
incubated with PHA, L-asparaginase, and one of the fol
lowing. L-glutamine, and L-asparagine plus L-glutamine.
After 48 hr incubation, thymidine-'H was added. DNA
synthesis in these groups was compared with controls con
taining PHA alone or PHA plus L-asparaginase. The up
take of thymidine-'H appeared augumented in these 3
groups (PHA, L-asparaginase plus L-asparagine; PHA,
(1 : 100) plus GPS
L-asparaginase plus L-glutamine; and PHA, L-asparagi
(ml/tube)0.125.022.539.0 nase, L-asparagine plus L-glutamine) as compared to that
of control treated with PHA plus L-asparaginase. The up
take of thymidine-'H in these 3 groups did not reach the
level of the PHA-treated control.
Table 3
Effect of CPS°and LAH A on the lymphocyte transformation
induced b\ PHA
(%')PÃŽI
Experi
ment
No.GPS
A(1:100)0.285.5
15.5
17.0
20.0
NT
NTPHA 50.0100)
12Experi
89.0
90.0
Table 5
Influence of L-asparagine and L-glutamine on the L-asparaginase
inhibition of PHA -induced lymphocyte transformation
(%'0.05NT46.573.5')(1:100)
(1:GPS plus LAHA
(mg/tube)1.0 LAHA
ment
No.LAHA
0.164.0
L-as
Test
paraginase,
ginase, L- ginase, L- L-asparagine,
cellNo.123PHA(cpm)23,83527,52539,314PHA,L-aspara
ginase
asparagine glutamine L-glutamine,
(cpm)3921,742717PHA,L-aspara
(cpm)4381,7711,157PHA,L-aspara
(cpm)6952,4551,630PHA,
(cpm)5953,8252,906
0.00140.0
1
2PHA
0
2.0
89.0
NT
14.5
48.0
90.0
NT
18.00.01 53.5
°
GPS, guinea pig serum; NT, not tested.
' %, blastic transformation rate.
52.5
NT
NT0.000165.0NT
NT
At time zero, L-asparaginase, L-asparagine, or L-glutamine was added
to each culture tube at a concentration of 2.0 i.u./tube, 0.05 mg/tube
and 3.0 mg/tube, respectively. Forty-eight hr after the start of culture,
2 ltd thymidine-'H were added to each culture tube and uptake of
thymidine-3H was measured 12 hr thereafter.
Table 4
L-Asparagine dependency and L-asparaginase sensitivity of PHA-induced transforming lymphocytes
PretreatmentNo
(cpm)Earle's
uptake
of preof L-as
of L-as
cell
incubation
paragine
de
paraginase
sen
No.565656565656Uridine-'H
(hr)00122448Test
pendency13.5-21.225.5-2,25.47.034.7-8.86.64.3-1
sitivity3.320.631.420.553.0
solution3705315326351,6351,5445,0285,10211,7769,333L-Asparagine
(0.05mg/tube)42041866S6211,7241.6636,7734,65112,5549,7399,706°11,934°L-Asparagi
(2.0i.u./tube)3584223655057716252,1181,4964,9575,177Values
treatmentPHAPHAPHAPHATime
' L-Asparagine content was 0.005 mg/tube.
770
CANCER
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VOL. 30
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L-Asparaginase and PHA-induced Lvmphocyte Transformation
DISCUSSION
E. coli L-asparaginase inhibited the lymphocyte trans
formation by PHA. The following mechanisms are con
sidered the inhibitory effect of this enzyme.
Acquisition of L:Asparaginase Sensitivity by Transform
ing Lymphocytes. The fact that the lymphocyte transfor
mation by PHA was inhibited not only by E. coli Lasparaginase and guinea pig serum but also by LAHA
suggests the involvement of the interference with L-asparagine metabolism as a possible mechanism.
The transforming lymphocytes showed L-asparaginase
sensitivity 12 hr after the initiation of culture, but they
did not require L-asparagine for their metabolism. This
might be due to the presence of L-asparagine in culture
medium. There are possibly 2 sources of L-asparagine in
tissue culture system. One is the fetal calf serum used in
this experiment which contained 7.80 ^g/ml L-asparagine
and the other is either red blood cells, platelets, or leuko
cytes. This discrepancy between the results on L-aspara
gine dependency and L-asparaginase sensitivity may imply
a mechanism other than specific disturbance of L-aspara
gine metabolism.
The Involvement of Glutaminase Activity in i.-Asparaginase in the Inhibition of PHA Lymphocyte Transforma
tion. Campbell et al. (8) reported that L-asparaginase
from E. coli had small but definite glutaminase activity.
Miller et al. (19) confirmed the presence of glutaminase
activity in this enzyme and found that the sera of the leukemic patients during L-asparaginase treatment contained
neither L-asparagine nor L-glutamine. The glutaminase
activity of our particular sample KW-020-1, Lot No. 1047,
was measured as about 4% of the L-asparaginase activity.
L-Glutamine as well as L-asparagine partially restored lym
phocyte transformation from the suppression with L-as
paraginase. Therefore, L-glutamine depletion in the tis
sue culture medium induced by L-asparaginase might be
a cause of the suppression.
PHA-stimulated lymphocytes in a metabolically acti
vated state synthesize proteins (2, 14, 24, 25) and nucleic
acids (9, 12, 26). For synthesizing of these materials, many
amino acids that are not essential for normal lymphocytes
may become essential for PHA-stimulated ones. The re
duced amount of exogenous L-asparagine and L-glutamine
in a tissue culture medium after treatment with L-aspara
ginase might become critically low for the process essen
tial for lymphocyte transformation, although adequate for
normal lymphocytes.
Other Possible Mechanisms of L-Asparaginase Inhibition
of PHA Lymphocyte Transformation. Another question is
whether L-asparaginase acts on the exogenous L-aspara
gine alone or on both exogenous and endogenous L-aspar
agine. Several unexplainable side effects of L-asparagi
nase treatment in patients with leukemia, such as hypofibrinogenemia (10, 11), hypocholesterolemia (11), liver
damage (11, 18), and myelosuppression (11) have been re
ported, although the highly purified, almost crystallized
MARCH
enzyme was used for the treatment. It seems difficult to
explain all of these side effects by the deprivation of exog
enous L-asparagine and L-glutamine by this enzyme. The
interference with the metabolism of endogenous L-aspar
agine and L-glutamine might be involved in these phe
nomena.
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CANCER
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VOL. 30
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Inhibitory Effect of l-Asparaginase in Lymphocyte
Transformation Induced by Phytohemagglutinin
Moriji Miura, Masami Hirano, Kumi Kakizawa, et al.
Cancer Res 1970;30:768-772.
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