The Effect of L-Asparaginase on the

Nucleic Acid Metabolism and Cell Cycle

of Human Leukemia Cells

By E. Fred Saunders

The effect of L-asparaginase on the contrast, thymidine labeling indices

cell cycle and nucleic acid synthesis decreased markedly to less than 50#{176}/o
of leukemic cells was studied in five of control by 6 hr. The changes in
children with acute lymphoblastic leu- labeling indices prior to changes in
kemia. Following an intravenous infu- mitotic indices indicated that L-aspa-
sion oi the drug, serial marrow sam- raginase blocked the entrance of cells
ples were obtained for buffy coat vol- into the DNA synthesis period of the
ume, mitotic index, and autoradio- cell cycle. Cells already in DNA syn-
graphic assessment of DNA and RNA thesis appeared to continue into mito-
synthesis using tritiated thymidine and sis. Uridine labeling indices decreased
tritiated uridine, respectively. A rapid progressively in all patients. Uridine
decline in buffy coat volume indicated uptake was inhibited equally in both
a lytic effect on lymphoblasts. There proliferative and nonproliferative
was a greater kill of proliferative blasts. Therefore, inhibition of RNA
(blasts in the cell cycle) than nonpro- synthesis by L-asparaginase was inde-
liferative (Go) leukemic cells. Mitotic pendent of the proliferative activity of
indices changed little until 24 hr; in the marrow.

L -ASPARAGINASE is an enzyme with chemotherapeutic activity against

human acute leukemia and some animal tumors. The drug acts primarily by
inhibiting protein synthesis in malignant cells deficient in L-asparagine syn-
thetase activity, and therefore, dependent on exogenous L-asparagine.”2 The
inhibition of protein synthesis is associated with inhibition of DNA and RNA
synthesis in certain experimental animals tumors.3’4 I studied the effect of
L-asparaginase on the cell cycle and nucleic acid metabolism of human acute
leukemia cells.

MATERIALS AND METHODS

The subjects were four girls and one boy ranging in age from 5 to 11 yr. All had
advanced acute lymphoblastic leukemia and had been treated with most available anti-
leukemic drugs. All patients were in relapse with 90% or more leukemic blasts in the
marrow. Previous antileukemic agents had been discontinued. No blood or platelet trans-
fusions, or drugs other than L-asparaginase were given during the study. Three of the
patients eventually achieved complete clinical and marrow remission. Informed consent
of the parents was obtained.

From the Department of Pediatrics, University of Toronto, and the Research Institute,
The Hospital for Sick Children, Toronto, Canada.
Submitted June 28, 1971; revised August 30, 1971; accepted October 6, 1971.
Supported by Grant 222 from the Ontario Cancer Treatment and Research Foundation.
E. Fred Saunders, M.D.: Assistant Professor of Pediatrics, Faculty of Medicine, University
of Toronto, and Staff Physician, Division of Hematology, The Hospital for Sick Children,
Toronto.

Blood, Vol. 39, No.4 (April), 1972 575

576 E. FRED SAUNDERS

z
I-)

A.

Fig. 1. Changes in buffy coat vol-

TIME (HOURS)
umes.

L-asparaginase (Farbenfabriken Bayer A. C.) produced from Escherichia coli was used.
Studies were done at the time of the first injection of L-asparaginase. Following a con-
trol bone marrow aspiration, 2000 U/Kg of the drug was given by intravenous drip
over a 30-mm period. Serial marrow samples were obtained from widely separated pelvic
sites during the subsequent 48 hr. Marrow was aspirated into a dry syringe and processed
for routine smears, buffy coat volume using a Wintrobe hematocrit tube, and mitotic
index using the acetocarmine suspension method.5 The mitotic index was obtained by
counting 10,000 nucleated cells per sample and expressed as the number of mitotic figures
per 1000 nucleated cells. The marrow needle was redirected at the same site and a heparin-
ized marrow sample obtained. One milliliter aliquots were incubated with thymidine-5-
methyl-3H (TdR3H) (0.36 Ci/mM), or uridine-5-3H (UR3H) (2.0 Ci/mM), in a concentra-
tion of 1 tCi/ml, for 1 hr at 37#{176}C. Autoradiographs were prepared using Kodak AR1O
stripping film; those with TdR3H were exposed 10 days and those with UR3H 14 days.
Background labeling was light and cells with five or more grains over the nucleus were
considered labeled. Two thousand leukemic blasts per sample were counted and the label-
ing indices expressed as a percentage.
Leukemic blasts may be classified morphologically as proliferative or nonprolifera-
tive.6 Dividing blast cells are large with fine nuclear chromatin; nondividing blast cells
are relatively smaller with coarse nuclear chromatin. Only the dividing cells incorporate
label during a 1 hr incubation with TdR3H. Both classes of cells incorporate UR3H. On
the autoradiographs, leukemic blasts were designated as proliferative or nonproliferative
depending on relative size and characteristics of the nuclear chromatin. Five hundred
blasts per sample were so classified. Mean grain counts were determined by counting 100
consecutive labeled blasts with five or more grains over the nucleus, per sample. The
significance of changes in labeling and mitotic indices, and grain counts was determined
by means of Student’s t test.

U
0
0
0

TIME (HOURS) Fig. 2. Changes in mitotic indices.

NUCLEIC ACID METABOLISM 577

U
0

z
U

Fig. 3. Changes in tritiated thymidine

labeling indices.
TIME (HOURS)

RESULTS

Changes in buffy coat volume are shown in Fig. 1. Two patients had marked
decreases in buffy coat volume by 6 hr and two had relatively gradual responses,
all reaching hypocellular levels by 48 hr. At that time the marrows consisted
of from 60% to 90% leukemic blasts; most of the remaining cells were small
lymphocytes. One patient started with a hypocellular marrow and changes
were not significant. Because leukemic blasts disappeared rapidly, determina-
tion of mitotic and labeling indices became difficult, and the study was dis-
continued at 48 hr.
Initial mitotic indices ranged from 5 to 15 per 1000 nucleated cells (Fig. 2).
They changed little during the first 6 hr, but then decreased steadily to very
low levels by 48 hr. TdR3H labeling indices of control marrows ranged from
4% to 18% (Fig. 3). In contrast to mitotic indices, labeling indices decreased
markedly by 6 hr, reaching almost zero by 48 hr in all cases. To compare mean
decreases in mitotic and TdR3H labeling indices for the five patients (Fig. 4),
means are expressed as a percentage of control values. By 6 hr, the mean
labeling index decreased to less than 50% of the control, whereas the mean
mitotic index was statistically unchanged. Thereafter, the two curves roughly
paralleled each other.
In two patients the size and labeling of only the proliferative population of
blasts in the marrow was followed. Initially, 23% and 31% of the marrow

>

z
0
U

z
U

Fig. 4. Comparison of mean changes

in mitotic and labeling indices (five
patients). TIME (HOURS)
578 E. FRED SAUNDERS

60

50-

U 40-

0 j T I I FIg. 5. Changes in tritiated uridine

0 12 24 36 48 labeling indices.
TIME (HOURS)

cells were large proliferative blasts. By 48 hr the proliferative population was

reduced to 10% and 8%, respectively. The remaining 90% of the blasts were
small nonproliferative cells. Labeling within the proliferative population de-
creased from control values of 51% and 32% to less than 10% by 48 hr.
The uptake of UR3H into leukemic cells is shown in Fig. 5. Labeling was
limited to the area over the cell nucleus. Initial labeling in 4 patients ranged
from 8% to 19%, and in the other was 56%. I cannot explain the high label-
ing in this instance. In all patients UR3H labeling indices decreased signifi-
cantly to an average of 25% of control values by 48 hr. The pattern of re-
sponse was variable; the patient with the highest initial labeling showed the
greatest response. In two patients the UR3H labeling indices were obtained
separately for the proliferative and nonproliferative blast populations. Initial
labeling of proliferative blasts was 32% and 41%, that of nonproliferative
blasts 5% and 8%. The relative decreases in labeling to about 30% of control
values by 48 hr were similar in both populations of cells.
Mean grain counts with both thymidine and uridine were not reproduce-
able. The pattern of change varied from patient to patient and could not be
interpreted statistically. However, the cells most heavily labeled with uridine
(>20 grains per cell) disappeared by 48 hr in each patient.

DISCUSSION

L-asparaginase is a useful drug in acute lymphoblastic leukemia in children

resulting in a 50-60% remission rate.7 Unfortunately, remissions are of short
duration. In vitro testing of drug sensitivity has not been useful in predicting
remission, probably because of rapid induction of L-asparagine synthetase
activity in some leukemic cells.8 Although all the patients had an initial drug
effect, only three achieved complete clinical and bone marrow remission. It
was impossible to detect any unique features of the patients, of the marrow
changes, or of the kinetic responses, which would allow one to predict the
patients who would eventually go on to remission.
The leukemic marrow consists of two populations of blasts, one proliferative
(cells in the cell cycle) and the other nonproliferative (resting blasts out of the
NUCLEIC ACID METABOLISM 579

cell cycle; C0 cells).6’9”0 The two leukemic cell populations are in equilib-
rium.11 Thymidine methyl-3H, a specific DNA precursor, is incorporated into
the cell nucleus only during the DNA synthesis (S) period of the cell cycle.
Decreases in thymidine labeling indices could have resulted from a relative
decrease in the total number of proliferative blasts in the marrow, and/or a de-
creased proportion of proliferative blasts in the S period of the cell cycle. The
data indicated that both events had occurred. By 48 hr most of the blasts in
the marrow were nonproliferative, indicating that L-asparaginase preferentially
killed proliferative cells. The decrease in the percentage of labeled cells within
the proliferative population indicated a decreased proportion of cells in S.
These effects must have been cell-cycle dependent.
However, the drug appeared to have another action independent of the cell
cycle. Nonproliferative blasts were killed. The marked decreases in buffy coat
volume by 6 hr after drug administration indicated a lytic effect on leukemic
cells which was too rapid to be cell-cycle dependent.
The drug reduced both thymidine labeling and mitotic indices, reflecting
decreased proportions of cells in the S and mitotic (M) periods of the cell cycle.
Though a reduction of cells in S was evident by 6 hr after the drug, changes in
cells in M were not detected until 24 hr. The data indicated that L-asparaginase
blocked the entrance of cells into the S period of the cell cycle. Cells already
in S appeared to complete DNA synthesis and go on to mitosis. The proportion
of cells in mitosis did not decrease until S became depleted of cells. The data
fit previously calculated DNA synthesis times of about 20 hr for leukemic
lymphoblasts.9 Because the number of marrow aspirations is limited in humans,
more detailed curves can not be drawn. In the presence of L-asparaginase, a
block in initiating DNA synthesis has been shown for Jensen sarcoma cells’2
and regenerating rat liver.’3 This effect is probably due to inhibition of protein
synthesis which is necessary for the initiation of DNA synthesis.’215 Pro-
tein synthesis may3’4” or may not’2”5”6 be necessary to maintain DNA syn-
thesis once initiated. However, various workers have studied different normal
or malignant tissues in experimental animals or tissue culture. As the mean
grain counts were uninterpretable, it was impossible to show whether DNA
synthesis rates had changed and, therefore, whether protein synthesis was
necessary to maintain DNA synthesis in human leukemia cells. Hydrocortisone
and prednisone block the initiation of DNA synthesis in human leukemic
lymphoblasts without inhibiting DNA synthesis already in progress.’7”8
Uridine-5-3H is a specific RNA precursor.’9 L-asparaginase gradually in-
hibited UR3H incorporation into RNA in all patients. RNA synthesis was
inhibited equally in proliferative and nonproliferative blasts. Therefore, this
effect of the drug was independent of the proliferative activity of the marrow.
Changes in mean grain counts could not be interpreted statistically. However,
the disappearance of heavily labeled cells reflected a decreased rate of RNA
synthesis. This study does not indicate which type of RNA was inhibited. In
a short incubation, human leukocytes incorporate UR3H into unstable, DNA-
dependent, nuclear RNA, which is partly ribosomal precursor RNA.2#{176}Mouse
lymphoma depleted of L-asparagine resulted in inhibition of all classes of
580 E. FRED SAUNDERS

RNA but especially ribosomal RNA.4 The inhibition of RNA synthesis is

probably directly due to the inhibition of protein synthesis. The effect of in-
hibition of protein synthesis on RNA fractions is complex and varies depend-
ing on the drug used, the cell system studied, and the duration of protein
starvation.21

ACKNOWLEDGMENT
I would like to thank Mrs. Larissa Besrutschenko for her excellent technical assistance.

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