[CANCER RESEARCH 33, 1628-1633,
July 1973]
The Effect of Methotrexate on Granulocytic Stem Cells and
Granulopoiesis1
William R. Vogler, Elizabeth S. Mingioli, and F. Ann Garwood
Division of Hematology and Medical Oncology, Department of Medicine. Emory University School of Medicine, A llanta, Georgia 30322.
SUMMARY
The effect of variations of methotrexate
schedules and
doses on granulocyte kinetics was investigated in C57BL mice.
Groups of animals were given single or repeated s.c. injections
of methotrexate
in doses of 60 or 120 mg/kg. Femoral
marrows were assayed daily from 1 to 12 days for total
cellularity, proliferating
and nonproliferating
granulocyte
pools, and the number of cells capable of forming colonies in
soft agar [colony-forming cells (CFC's)]. Leukocyte counts
and differentials were performed daily. Following a 60-mg
dose of methotrexate
per kg there was a reduction in
proliferating pool to 43% of control on Day 2 and a 2.3-fold
rise in CFC's/10s marrow cells on Day 3, returning to initial
value by Day 6. Repeating the dose of methotrexate on Day 6
resulted in a similar response. Repeating methotrexate on Day
3, at a time when the CFC's were elevated, resulted in a fall in
the proliferating pool to 8% of the control value and 3.4-fold
rise in CFC's/10s marrow cells. There was a more pronounced
reduction in total cellularity and circulating leukocytes than
when the second dose was given on Day 6. When 120 mg of
methotrexate per kg were given followed by 60 mg on Day 3
there was a ninefold increase in CFC's/IO5 marrow cells that
persisted accompanied by a marked and prolonged reduction
in marrow cellularity.
These results document
the effect of scheduling of
methotrexate on granulocyte stem cell kinetics. This knowl
edge will permit the design of less toxic chemo therapeutic
schedules and can serve as a model to study the molecular
events occurring in granulopoiesis after perturbations of the
marrow.
INTRODUCTION
Certain classes of drugs used in cancer chemotherapy are
effective against proliferating cells. Cells not in mitotic cycle
(resting cells) are not affected by these drugs. Bruce et al. (4)
have shown by the spleen colony technique that, under normal
circumstances, only a fraction of marrow stem cells is
proliferating.
However, following a perturbation
of the
marrow, a greater proportion
of stem cells enters into
proliferation
(5). Chemotherapeutic
agents active against
'Supported by Research Grants T-567 from the American Cancer
Society and CA 11692 from the NIH.
Received December 29, 1972; accepted March 22, 1973.
1628
proliferating cells, given at a time when a greater fraction of
stem cells is proliferating, should result in a greater percentage
kill and cumulative toxicity should ensue. However, the
administration of a drug after return of the stem cell to the
resting state should result in no additional toxicity. With the
development of the agar cloning technique a method is
available for determining quantitatively an apparent granulocytic (committed) stem cell (2,11, 14). This study measures
the effect of single and repeated doses of methotrexate, a drug
active against proliferating cells (1), on the granulocytic stem
cell compartment
and correlates these changes with the
marrow cellularity, proliferating and nonproliferating marrow
granulocyte pools, and circulating leukocytes.
MATERIALS
AND METHODS
Details of the agar cloning technique used in these studies
have been described previously (13). Here the technique is
briefly summarized. Pairs of C57BL mice were subjected to
single or repeated s.c. injections of methotrexate given in doses
of 60 or 120 mg/kg. One to 12 days following the last
methotrexate dose the mice were anesthetized with sodium
pentobarbital, blood counts and smears were obtained from
peripheral blood (tail or retrorbital), and both femurs were
removed aseptically. The cells were expressed from the shaft
of 1 femur of each mouse by vigorous and repeated aspirations
through a 25-gauge needle with 1.5 or 2 ml of modified
McCoy's medium (10). A pooled cell suspension from each
pair of animals containing 2.5 X IO4 to 10s cells per 0.7 ml of
medium plus 0.3% agar was pipetted into quadruplicate
35-mm Falcon Petri dishes over feeder layers of medium and
agar containing either 15% by volume of kidney supernatant
(obtained from medium incubated 7 days with 8- to 10-day-old
minced mouse kidneys: 12 kidneys per 50 ml medium) or
2.5% by volume of stimulating factor isolated from the urine
of leukemic patients. Plates were incubated at 37°in moist
7.5% C02 in air for 7 days. Following incubation colonies
containing a minimum of 16 cells were counted using a
stereoscopic dissecting microscope. Most colonies contained
100 to 300 cells each. From the 2nd femur of each mouse the
cells were expressed onto glass slides, and smears were made
and stained with Wright's stain. Three hundred cell differential
counts were done on each specimen. Differential counts of
marrow aspirates contained on the average 7% more granulocytes than the pooled suspensions. Because of this small
difference no corrections were made.
CANCER RESEARCH
VOL. 33
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Methotrexate
and Granulopoiesis
The number of points measured per experiment was limited
by the number of plates of colonies to be counted. In each
experiment a control plus 3 to 4 experimental points (2 mice
per point) were the maximum that could be counted at 1'time.
Thus the data presented below are a composite of 18
experiments. For most of the 12 experimental points (days
after the 1st dose of methotrexate) the data are derived from 2
to 4 experiments. Because of variation between experiments in
the numbers of colonies per 10s cells in the controls, the
values for CFC's2 are expressed as a percentage of the control.
The means and the standard errors of all of the observations
for a particular point are calculated. Student's t test was used
**
C
for a particular point to test the hypothesis that the value of a
given variable is no different than 100. The level of
significance chosen for the colony count data was 10%.
The proliferating marrow granulocyte pool consisted of
myeloblasts, promyelocytes, and myelocytes. The nonproliferating pool consisted of metamyelocytes, bands, and segmented
neutrophils. Although it is generally acknowledged that only
some myelocytes are capable of division, these could not be
morphologically distinguished from nondividing myelocytes
and all myelocytes were included in the proliferating pool.
RESULTS
Colony counts of quadruplicate plates for a particular
experimental point were similar. Usually, the S.E. of the
means varied by less than 5%.
DAYS
Chart 2. Effect of methotrexate (MTX) on the proportion of CFC's
per 10s marrow cells. Bars, S.E.'s of the means of 2 or more
experiments; top, methotrexate (60 mg/kg) given on Day 0; middle,
methotrexate (60 mg/kg) given on Days 0 and 3; bottom, methotrexate
(60 mg/kg) given on Days 0 and 6.
Variation in the plating efficiency of CFC's in control plates
occurred between experiments. Pooled marrow samples from 2
mice in each of 10 experiments gave a mean of 406.2 ±80.9
colonies per 10s marrow cells.
Charts 1 and 2 are summary charts of our total experience
showing the effect of methotrexate on colony formation.
Chart 1 gives the effect of single and multiple doses of
methotrexate (60 mg/kg) on the absolute number of CFC's per
femur. The number appeared to
experiments following a single
higher than the control value
methotrexate on Day 3 resulted
CFC's reaching a nadir (44 ±4%
increase by Day 3 in 2 of 3
dose and was significantly
on Day 6. Repeating the
in a significant reduction in
of control) on Day 6 before
recovery took place. Repeating the methotrexate on Day 6
resulted in a transient nonsignificant reduction on Day 7 (74 ±
12% of control) followed by recovery by Days 9 and 10. Thus,
it would appear that there is recruitment or activation of
CFC's following methotrexate administration and when the
246
DAYS
Chart 1. Effect of methotrexate (MTX) on absolute number of
CFC's per femur. Bars, S.E.'s of the means of 2 or more experiments;
top, methotrexate (60 mg/kg) given on Day 0; middle, methotrexate
(60 mg/kg) given o'n Days 0 and 3; bottom, methotrexate (60 mg/kg)
given on Days 0 and 6.
2The abbreviation used is: CFC's, colony-forming cells.
dose is repeated at the time of maximum activation a greater
kill of CFC's occurs.
A different pattern was observed when looking at the
proportion of marrow cells capable of in vitro colony
formation. As shown in Chart 2, methotrexate administration
resulted in a rise in CFC's after each dose. When the doses of
methotrexate were given 6 days apart (Chart 2. bottom) the
response to the 2nd dose was similar to the 1st, falling toward
JULY 1973
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1629
W. R. Vogler, E. S. Mingioli, and F. A. Garwood
the control value 4 to 5 days later. When methotrexate doses
were administered 3 days apart (Chart 2, middle) the
proportion of CFC's remained above the control value for 6
days.
The effect of single or multiple doses of methotrexate on
the marrow total cellularity and granulocyte pools, the
peripheral blood counts, and the CFC's is shown in Table 1.
The relationship of the changes in marrow granulocyte pools
to the proportion of cells capable of forming colonies is
illustrated in Charts 3 to 5. Following a single dose of
methotrexate (Chart 3), there is a rapid fall in the proliferating
pool reaching a nadir of 43 ±3% of control on Day 2. A
2.3-fold rise in CFC's/10s marrow cells occurred on Day 3. By
Day 6 the values had returned to the base line and oscillated
about this for the remainder of the period of observation.
Repeating methotrexate on Day 6 (Chart 4) resulted in a fall
in marrow granulocyte pools similar to the 1st dose. There was
a 2nd 3.6-fold rise in CFC's/10s marrow cells. The nadir of the
proliferating pool was similar to that observed after the 1st
dose. However, when the dose of methotrexate were given on
Days 0 and 3, at a time when the proportion of CFC's was
elevated, the marrow proliferating pool fell to 8.8 ±0.1% of
the control value on Day 6. The CFC's/10s marrow cells
following the 2nd dose of methotrexate gave a 2nd rise
exceeding the control by a factor of 3.4, before returning to
control values 6 days after the 2nd dose.
Table 1
Effect of singleand multiple doses of methotrexate on marrow, blood, and CFC's
Marrow
cellularityDay
Total
Na
Blood
marrow cellsProliferating
control of
Cells X 10«•% CFC's/femurCFC's/10s
% control
N
poolsNon-proliferating
(ceils X 106 )Granulocyte
(cells X IO6)WBC/cu
mm
Neutrophils/cu mm
Methotrexate Day 0 (60 mg/kgj
0
1
2
3
4
5
6
7
8
9
10
11
12
10
3
2
3
2
2
3
2
2
2
2
1
3
18.11+ 2.02o
11.02±6.36e'
8.40 ±1.34C
9.32 ±0.94d
11.50+ 2.07
18.81 ±.8.05
17.98 ±3.60
21.20 ±-0.40
20.32 ±1.19
16.45 ±4.92
25.19 ±4.82
22.00 ±
0
18.75 ±1.58
100 ±O
102+ 14
88 ±2
190 ±
53
148±11
112+ 13
174 ±IO"1
97± 4
132+ 28
73 ±20
91 ±9
55 ±0
104 ±27
100 ± 0
114+ 28
148± 26
233 ±S2e
136± 12
104± 26
157 ±43
101± 10
137± 4e
88 ±18
89+ 15
67 ± 0
104 + 22
18
5
4
5
3
3
5
4
4
3
3
2
5
3.21 + 0.25
1.89 ±0.05e
0.09C
1.39 ±
0.21e
2.10 ±
0.54e
2.18 ±
2.69 ±1.08
4.81 ±0.61d
0.08
3.49 ±
2.99 + 0.18
3.27 ±0.35
0.09e
3.71 ±
3.14 + 0.41
3.52 ±0.36
8.59 ±
0.80
5.68±1.13d
4.74 + 0.77e
0.17e
3.81 ±
4.61 1.13e
5.58 2.41
7.05 1.44
10.05 0.29
10.94 1.21
7.76 194
11.04 2.77
13.10 0.18e
8.74 1.56
12,173 ± 890
6,193 + 1,569e
8,097 1,253e
530e
7,009
8,633 1,921
8,936 2,471
15,960 1,848e
11,375 1,698
10,656
954
8,692 2,134
12,233
586
10,388 2,979
3,578e
18,526 ±
1,748 + 199
1,769 ± 563
1,814 ± 263
1,422 + 153
2,080 ± 143
3,183 ± 835
2,618 + 319e
1,716 ± 252
1,588± 196
1,351 ± 485
2,068 ± 438
3,536 + 339e
3,400 ±1,174
Methotrexate Days 0 and 3 (60 mg/kg)
4
5
6
7
8
9
10
11
12
7.51±3.24d
5.23 2.37e
2.73 0.25e
17.87 7.59
17.78 O
18.60 2.83
19.53 1.61
23.70 ±1.77e
20.80 ±O
86 ±28
66 ±26
44+ 4e
136 ±27
207+ 0
105 ±2
97 ±48
96 ±46
69 ±0
178 ± 36e
196 ±50
247 85
343 127
213
0
110 24
113 54
80 + 29
66 ± 0
1.17±0.68d
0.69+ 0.19e
0.29 + 0.00e
301 ±1.01
2.28+ 0.10e
3.14+ 0.57
3.73+0.34
3.60 + 0.39
3.38 ±
0.20
3.52
2.53
0.93
8.05
8.28
6.81
8.01
8.61
8.87
1.10e
0.82e
0.04e
2.53
0.07
0.77
0.78
0.47
0.49
7,624
4,958
3,680
9,545
6,689
22,167
12,800
15,825
19,200
482e
982e
261e
2,853
1,90o11
2,353e
1,134
2,140
495e
2,223 ± 829
1,942 ± 515
1,589
738
2,272
686
2,259
271
419e
2,559
423e
2.604
462e
3,353
2,084
489
Methotrexate Days 0 and 6 (60 mg/kgj
1.2810.21
±
7891011122222248.45
1.099.80
±
1.446.90
±
0.7313.17
+
3.5717.64+
±
1.7974
0.25e1.49
±
12190+
0.10e2.12
±
2e361
0.19e1.
±
63221
±
85182+
±
0.67e3.83
16 ±
28137
6191
±
0.804.04
±
40163
±
16175
±
0.15e4.45
±43162+
±513433371.95 ±
1288+3198
±
0.73e5.30
±
+6,531
0.68e5.27±
+
±4,095
0.90"*2.64
+7,717
0.47e4.85
+
±12,923
0.94e8.86
±
±13,159
+118e931e712e621e1,7524,3616622251
± 403
±0.517,368
0 Number of observations.
b Mean ±
S.E. of 2 or more experiments.
e Varies significantly from control values. Level of significance, 1%.
d Varies significantlyfrom control values.Levelof significance,5%.
e Varies significantly from control values. Level of significance, 10%.
1630
CANCER RESEARCH VOL. 33
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W. R. Vogler, E. S. Mingioli, and F. A. Garwood
120 mg of methotrexate
per kg and the effect on the CFC's,
proliferating granulocyte pool, and total marrow cellularity is
expressed as percentage of control. In contrast to the single
60-mg/kg dose, the CFC's/10s marrow cells showed an initial
10% drop followed by a similar rise reaching a peak on Day 3
that was significantly higher than control values (p < 0.01).
The total cellularity and proliferating pool fell significantly (p
< 0.01) to approximately 40% of control value on Day 2 and
both recovered by Day 5. When a 2nd dose of methotrexate
(60 mg/kg) was given on Day 3 (Chart 7), there was a 9-fold
rise in CFC's/10s marrow cells (a highly significant difference
(p < 0.01)) that persisted, accompanied by a marked and
prolonged significant (p < 0.01) reduction in proliferating
pool and marrow cellularity.
DISCUSSION
on CFC's.
The peak occurred
at 3 days and was
followed by a rapid decline. The fact that these peaks
preceded increases in proliferating pools and marrow recovery
is in keeping with the concept that the CFC is a granulocyte
precursor (8).
When a 2nd dose of methotrexate was given at a time when
the marrow was perturbed (Day 3), and at the peak of CFC
activity when more cells should be proliferating, there was an
initial 70% drop followed by a sustained rise in CFC's. As
expected, a marked fall in proliferating pool and marrow
cellularity occurred. A much greater proportion of cells in the
marrow were CFC's, although the total number of CFC's was
reduced. This resulted in delayed recovery.
dose of methotrexate on Day 6, at a time
was much less perturbed, much less toxicity
the pattern of response of the CFC's was
By giving the 2nd
when the marrow
was observed and
similar to a single
dose.
Somewhat similar changes in relative proportion
These experiments illustrate the varied effects of different
doses and frequency of administration of methotrexate on
granulocyte compartments and CFC's. When methotrexate was
given at a time when the marrow was under normal
homeostatic conditions, the changes observed in compart
ments and CFC activity were dose related. The smaller dose
resulted in less marrow toxicity and earlier recovery. The
larger dose initially reduced CFC's at Day 1 followed by a
peak on Day 3 and gradual decline to control values by Day 6.
In contrast, the smaller dose resulted in a prompt rise in CFC's
at Day 1, although a lag in the curve suggests some inhibitory
900
800
700
600
500
CFC'S / IO5
400
Cells
Proliferating
Pool
Total
Cellularity
•¿
300
-
200
120
100
100
•¿I
N'r\A
'
//-
-v**-**1
^
1
MIX
0
effect
1
1
60to20
180
n
MIX
10
DAYS
of CFC's
were observed by Brown and Carbone (3) following a single
injection of cyclophosphamide using the in vitro technique
with mouse marrow. They observed an initial decline followed
by almost a 4-fold increase in CFC's on Day 3 with a return to
control levels by Day 6. Extending their observations
additional days they noted a secondary fall in CFC's on Days 8
to 10 that was as severe as the initial decline, before recovery
on Day 11. We saw a significant decrease from the control
value on Day 9 following a 60-mg dose of methotrexate per
kg. One might attribute these changes to an injury-induced
increase in amplitude of the normal oscillations about the base
line similar to that described by Morley and Stohlman (9).
They produced cyclic neutropenia in dogs by mild bone
marrow depression induced by daily cyclophosphamide.
King-Smith and Morley (7) proposed a model of granulopoiesis
in which the neutrophil count controlled production by a
feedback loop acting on the stem cell. However, Chervenick
and Boggs (6) presented evidence, using the spleen colony
assay technique, suggesting that stem cell compartment
depletion was the factor most closely correlated with stem cell
proliferation. Undoubtedly, the mechanism of granulocyte
regulation is complex and probably involves several feedback
loops. We have found factors in the urine of methotrexatetreated mice that stimulate and inhibit colony formation
suggesting that humoral mechanisms are involved (13).
Recently, Stohlman (12) has reviewed the relationship of
colony-stimulating factor and myelopoiesis. Obviously, more
information is necessary to clarify the factors involved in
marrow recovery. However, from the work of Brown and
Carbone (3) and our studies reported here, it is likely that an
in vitro cloning technique that is applicable to human studies
may be of value in improving treatment schedules.
In addition, this method of marrow perturbation serves as a
model for investigating the regulatory factors involved in
granulopoiesis.
Chart 7. Effect of 120 mg methotrexate (MTX) per kg followed by
60 mg/kg on Day 3 on the CFC's, proliferating pool, and total marrow
cellularity. The values for Days 0 to 3 are from Chart 6. The remaining
data are from a single experiment in which quadruplicate plates of
CFC's varied significantly (p < 0.01) from control plates on Days 5, 7,
ACKNOWLEDGMENTS
and 9, and total cellularity and proliferating pool varied significantly
from control values (p < 0.01).
The authors express their appreciation to Dr. Yick-Kwong Chan for
statistical assistance.
1632
CANCER
RESEARCH
VOL.
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33
Methotrexate
REFERENCES
1. Borsa, J., and Whitmore, G. F. Cell Killing Studies on the Mode of
Action of Methotrexate on L Cells in Vitro. Cancer Res., 29:
737-744, 1969.
2. Bradley, T. R., and Metcalf, D. The Growth of Mouse Bone
Marrow Cells in Vitro. Australian J. Exptl. Biol. Med. Sci., 44:
287-300, 1966.
3. Brown, C. H., Ill, and Carbone, P. P. Effects of Chemotherapeutic
Agents on Normal Mouse Bone Marrow Grown in Vitro. Cancer
Res.. 31: 185-190,1971.
4. Bruce, W. R., Meeker, B. E., and Valeriote, F. A. Comparison of
the Sensitivity of Normal Hematopoietic and Transplanted
Lymphoma Cells to Chemotherapeutic Agents Administered in
Vivo. J. Nati. Cancer Inst., 37: 233-244, 1966.
5. Chen, M. G., and Schooley, J. C. Recovery of Proliferative
Capacity of Agar Colony-forming Cells and Spleen Colony-forming
Cells following Ionizing Radiation or Vinblastine. J. Cellular
Physiol., 75: 89-96, 1970.
6. Chervenick, P. A., and Boggs, D. R. Patterns of Proliferation and
Differentiation of Hematopoietic Stem Cells after Compartment
Depletion. Blood, 37: 568-580,1971.
JULY
and Granulopoiesis
1. King-Smith, E. A., and Morley, A. A. Computer Stimulation of
Granulopoiesis: Normal and Impaired Granulopoiesis. Blood, 36:
254-262,1970.
8. McCulloch, E. A. Differentiation of Hematopoietic Stem Cells.
Xllth Congress of the International Society of Hematology, New
York, 1968, pp. 260-271.
9. Morley, A. A., and Stohlman, F., Jr. Cyclophosphamide-Induced
Cyclical Neutropenia. New Engl. J. Med., 282: 643-646, 1970.
10. Pike, B. L., and Robinson, W. A. Human Bone Marrow Colony
Growth in Agar-Gel. J. Cellular Physiol., 76: 77-84,1970.
11. Pluznik, D. H., and Sachs, L. The Cloning of Normal "Mast" Cells
in Tissue Culture. J. Cellular Comp. Physiol., 66: 319-324, 1965.
12. Stohlman, F., Jr. Colony-Stimulating Factor and Myelopoiesis.
Blood, 39: 727-732, 1972.
13. Vogler, W. R., Mingioli, E. S., Garwood, F. A., and Smith, B. A.
Granulopoietic Stem Cell Regulators in Murine Urine: Alterations
in Activity after Methotrexate. J. Lab. Clin. Med. 79: 379-387,
1972.
14. Wu, A. M., Siminovitch, L., Till, J. E., and McCulloch, E. A.
Evidence for a Relationship between Mouse Hemopoietic Stem
Cells and Cells Forming Colonies in Culture. Proc. Nati. Acad. Sci.
U. S., 59: 1209-1215,1968.
1973
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1633
The Effect of Methotrexate on Granulocytic Stem Cells and
Granulopoiesis
William R. Vogler, Elizabeth S. Mingioli and F. Ann Garwood
Cancer Res 1973;33:1628-1633.
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