[CANCER RESEARCH 32, 2459-2462, November 1972]
Effects of Methotrexate and Cytosine Arabinoside on Polyamine
Metabolism in a Mouse L1210 Leukemia
Diane H. Russell
Baltimore Cancer Research Center, Laboratory of Pharmacology, National Cancer Institute, NIH, Baltimore, Maryland 21211
SUMMARY was purchased from Calbiochem, Gaithersburg, Md. NSD-1055
(4-bromo-3-hydroxybenzyloxyamine dihydrogen phosphate)
The effects of methotrexate and cytosine arabinoside on was a gift from Smith and Nephew Research Ltd., Welwyn-
polyamine metabolism were studied in a rapidly proliferating Garden City, Hertz, United Kingdom. DBA/2 mice with
leukemic tumor (LI210) in C57BL/6 X DBA/2 (hereafter L1210 ascites tumors were obtained from Microbiological
called BDP!) mice. Methotrexate, the most effective Associates, Bethesda, Md. These mice were used to carry the
antileukemic drug in terms of increasing the median survival tumor strain. The studies reported herein were performed on
time also was the most effective, both in inhibiting the the spleens of BDF, mice that had been given injections of
enzymes in the polyamine biosynthetic pathway and in 1 X IO6 cells from the carrier strain. The spleen is rapidly
decreasing the accumulation of polyamines by the tumor cells. invaded by the ascites tumor cells, and thus it accurately
reflects the tumor activity throughout its time course
(Microbiological Associates, personal communication). Protein
was determined by the method of Lowry et al. (5), with
bovine serum albumin as the standard. There was a constant
Much evidence indicates that one of the earliest events in
ratio between the protein content and wet weight of the
any growth system is an increased synthesis and accumulation
spleen in all age groups.
of polyamines (1, 7, 8, 12, 14). This is true for plant, bacterial,
Enzyme Assays. For enzymatic assays, the spleens of
and mammalian systems (10, 15). It is also true for systems
tumorous mice or controls were excised, chilled on ice, rinsed
exhibiting both normal and neoplastic growth (9, 12). Because
in 0.9% NaCl solution, blotted, weighed, and immediately
polyamine biosynthesis is one of the earliest detectable events,
some workers have used the activity of ornithine decarboxyl- homogenized in 5 volumes of cold 0.05 M sodium phosphate
buffer, pH 7.2, containing 1 mM dithiothreitol. The
ase, the 1st enzyme in the polyamine biosynthetic pathway, as supernatant preparation, after 20,000 X g centrifugation, was
an indicator of stimulation after administering a growth-
used in the enzymatic assays.
producing hormone or a mitogenic agent (4). Perhaps it is Assay for Ornithine Decarboxylase Activity. Ornithine
timely to consider the possibility of using the early indications decarboxylase activity was determined by the liberation of
of polyamine biosynthesis and accumulation in tumors as 14CO2 from carboxyl-14C-labeled ornithine, as previously
indices of the growth rates of tumors and also as indices of the described (12, 13).
effectiveness of antileukemic agents. Assay for Putrescine-dependent S-Adenosyl-L-methionine
In this study, we report on 2 antileukemic agents that are Decarboxylase Activity or Spermidine Formation. Enzyme
used in the clinic to treat leukemia. The most effective of activity was determined by measuring the liberation of ' 4CO2
these drugs in terms of antileukemic properties is metho from S-adenosyl-L-methionine-carboxyl-14C, as previously de
trexate, and indeed methotrexate dramatically inhibits both scribed (6). The evolution of 14C02 from 5-adenosyl-L-
the biosynthesis and accumulation of polyamines in mouse methionine-carboxyl-14C, when putrescine is present as an
L1210 leukemia. Cytosine arabinoside, which is less effective,
acceptor for the propylamine moiety from decarboxylated
inhibits the biosynthesis and accumulation of the polyamines S-adenosylmethionine, is a valid measure of the rate of
to a lesser extent.
spermidine synthesis (2, 3). Incubation mixtures consisted of
150 juM S-adenosyl-L-methionine-carboxyl-14C; 50 mM
MATERIALS AND METHODS sodium phosphate buffer, pH 7.2; 50 /uM pyridoxal phosphate;
2.5 mM putrescine; and 0.05 to 0.10 ml of enzyme
Materials.DL-Ornithine-l-I4C (3.31 mCi/mmole), putres- supernatant solution prepared as described above, to make a
cine-l,4-14C dihydrochloride (11.3 mCi/mmole), spermidine- total volume of 0.2 ml.
14C trihydrochloride (10.7 mCi/mmole), spermine-14C The concentration of S-adenosyl-L-methionine used in these
tetrahydrochloride (9.9 mCi/mmole), and S-adenosyl-L- assays was nonsaturating. However, in those experiments in
methionine-carboxyl-14C (7 mCi/mmole) were obtained which a saturating level was used, the values obtained were
from New England Nuclear, Boston, Mass. The hydrochloride proportionate to those reported here.
salts of putrescine, spermidine, and spermine were obtained All values were corrected against a heated reaction assay,
from Sigma Chemical Co., Boston, Mass. Pyridoxal phosphate and NSD-1055 (4-bromo-3-hydroxybenzyloxyamine di
hydrogen phosphate), an inhibitor of pyridoxal phosphate-
Received May 30, 1972; accepted August 3, 1972. dependent enzymes, was also to corroborate the blank values.
NOVEMBER 1972 2459
Diane H. Russell
Assay for Spermidine-dependent S-Adenosyl-L-methionine Ornithine Decarboxylase Activity. Ornithine decarboxylase
Decarboxylase or Spermine Formation. The evolution of activity is dramatically elevated in the tumor-inoculated mice
14C02 from S-adenosyl-L-methionine-carboxyl-14C, when by Day 3 (Table 1). By Day 6, it is nearly 7-fold that found in
spermidine is added as the acceptor for the propylamine the normal spleen, and it remains elevated by Day 8. When
moiety from decarboxylated S-adenosyl-L-methionine, is a either methotrexate or cytosine arabinoside is given daily to
valid measure of the rate of spermine synthesis (3, 11). the tumor-inoculated mice, ornithine decarboxylase activity
Therefore, I4C02 evolution was used routinely to estimate on Days 3, 6, and 8 is depressed generally. There is some
the spermine synthesis activity. Incubation mixtures consisted indication that by Day 8 it may be escaping from the
of 150 jUM S-adenosyl-L-methionine-carboxyl-14C; 50 mM inhibition. When only methotrexate or cytosine arabinoside is
sodium-potassium phosphate buffer, pH 7.2; 50/aM pyridoxal administered, there is a general increase in the level of
phosphate; 5 mM spermidine; and 0.05 to 0.10 ml of enzyme ornithine decarboxylase activity. The amount of stimulation
supernatant in a total volume of 0.2 ml. appears to be fairly constant at all of the times assayed.
Determination of Endogenous Concentrations of Putrescine, Putrescine-dependent S-Adenosyl-L-methionine Decarboxyl
Spermidine, and Spermine. The endogenous concentrations of ase Activity. In contrast to the increased activity of ornithine
putrescine, spermidine, and spermine of the spleens were decarboxylase activity detectable in the tumor cells by Day 3,
examined after extraction of the amines into alkaline putrescine-dependent 5-adenosyl-L-methionine decarboxylase
1-butanol, separation by high-voltage electrophoresis (80 activity is not elevated at that time (Table 2). However, the
V/cm), and staining with acid ninhydrin, as previously activity has increased by Day 6 and remains elevated by Day 8.
described (10). Tracer amounts of radiolabeled amines were Methotrexate administration decreases the level of 5-adenosyl-
added to some tubes in each experiment, and the recovery rate L-methionine decarboxylase activity by Day 3 to less than
for each amine was determined as previously described (10). one-half that found in the tumorous animals alone. This
depression in the enzyme activity is also observed on Day 6,
RESULTS but the enzyme activity appears to escape the inhibitory
The drug studies reported herein were performed Table 2
simultaneously to avoid a change in the virulence of the The effects of methotrexate and cytosine arabinoside on putrescine-
L1210 tumor cells, which is known to occur when studies dependent and spermidine-dependent S-adenosyl-L-methionine
continue over a prolonged period of time. decarboxylase activities at various times
after tumor inoculation
Groups of 10 BDF, male mice were inoculated with IO6 L1210
Table 1 ascites cells and controls were inoculated with 0.9% NaCl solution. The
The effects of methotrexate and cy tosine arabinoside on ornithine spleens were assayed for putrescine-dependent and spermidine-
decarboxylase activity at various times after tumor inoculation dependent S-adenosyl-L-methionine decarboxylase activities on the 3rd,
Groups of 10 BO!-', male mice were inoculated with IO6 L1210 6th, and 8th days after the tumor inoculation. The enzyme activities
were assayed by measuring the evolution of ' 4CO2 from 5-adenosyl-L-
ascites cells, and controls were inoculated with 0.9% NaCl solution. The
methionine-carboxyl-'4C as described in "Materials and Methods."
spleens were assayed for ornithine decarboxylase activity on the 3rd,
6th, and 8th days after the tumor inoculation. Ornithine decarboxylase Groups receiving drugs were injected i.p. with either cytosine
activity was assayed by measurement of the evolution of ' 4CO2 from arabinoside (23 mg/kg) or methotrexate (2 mg/kg) on Days 1 through
DL-ornithine-1-1 4C, as described in "Materials and Methods." Groups S
9. Each value represents the mean Â± .E. of duplicate determinations of
receiving drugs were given i.p. injections of either cytosine arabinoside at least 4 separate pools of spleens.
(23 mg/kg) or methotrexate (2 mg/kg) on Days 1 through 9. Each value
represents the mean Â± .E. of duplicate determinations of at least 4
separate pools of spleens. activities(pmoles'4COJ30min/mg
activity (pmoles I4CO2/30
13198 21 92 Â±
42Â°199 inoculatedTumor Â±30
Â± 23b8638 182
plusmethotrexateTumor 10115 16b 72
23Â°83Â± 11184 15b 78
inoculated llb71 18155 29 111
arabinosideMethotrexate 13184Â± inoculated
pluscytosine 16190 24 119
22a73Â± arabinosideMethotrexate 22316 33 116
onlyCytosine 1059 57b15651b 247
Â±897 onlyCytosine 15164 27 110
12"99 2189 29 125
arabinoside onlyDay36X3683683ft8368Ornithine 9175 12b 76
12692 arabinoside onlyDay368368368368368S-Adenosyl-L-methioninedecarboxylas
25140 30 133
Â±15b 2678 34 114
10b 32 8b
a Data differ from control (p < 0.001). " Data differ from control (p < 0.05).
b Data differ from control (p < 0.05). b Data differ from control (p < 0.001).
2460 CANCER RESEARCH VOL. 32
Polyamine Metabolism in a Mouse LI210 Leukemia
Concentrations ofputrescine, spermidine, and Â¡perniinein spleens of normal, tumor-inoculated,
and drug-treated mice
Groups of 10 BDF, mice were inoculated with IO6 L1210 ascites cells and the controls were
inoculated with 0.9% NaCl solution. The spleens were assayed for endogenous levels of polyamines on the
3rd, 6th, and 8th days after tumor inoculation. The spleens were pooled (minimum of 3 pools), and the
endogenous levels of the polyamines were assayed by extraction of amines into alkaline butano! and
separation of high-voltage electrophoresis, as described previously (11). Data are presented as the
mean Â± .E. for duplicate determinations of at least 3 pools of 10 separate spleens.
Concentrations (nmoles/mg protein) of
Experimental group Day Putrescine Spermidine Spermine spermine ratio
Â±0.1314.2 Â± 1.614.3 Â± 1.80.922.214.171.124.126.96.36.199.31.41.11.01.21.01.11.0
" Data differ from control (p < 0.05).
0 Data differ from control (p < 0.001).
effects of methotrexate by Day 8. The same general pattern is methionine decarboxylase is elevated above controls on Days 3
true after the administration of cytosine arabinoside, except and 6 and is depressed by Day 8.
that S-adenosyl-L-methionine decarboxylase activity is not as Survival Time of the Various Groups. All of the mice given
depressed by Day 3. Its level is about 80% that found in the injections of tumor alone were dead within 9 days of tumor
tumorous mice. Actually, by Day 8, the S-adenosyl-L- inoculation. The group inoculated with tumor plus metho
methionine decarboxylase activity in the tumorous mice given trexate had a median survival time of 19 days. Those injected
injections of cytosine arabinoside is higher than the level of with tumor plus cytosine arabinoside had a median survival
activity found in the tumorous mice that were not given time of 16 days. Neither cytosine arabinoside nor metho
injections. This is probably a reflection of the inability of the trexate, when administered to normal animals, caused any
antileukemic agent to continue to suppress tumor growth. mortality rate if given for only the 9-day period. However,
When either methotrexate or cytosine arabinoside is adminis when the injections of cytosine arabinoside or methotrexate
tered to nontumorous animals, the level of putrescine-stimu- were carried further, methotrexate proved very toxic indeed,
lated S-adenosyl-L-methionine decarboxylase is depressed and the mice died after 20 days on the drug regimen. Cytosine
slightly on Days 3 and 6 and is depressed markedly by Day 8, arabinoside given for the same period of time caused no
indicating that these agents do suppress normal activity. mortality.
Spennid ine-dependent S-Adenosyl-L-methionine Decarbox The Effects of Methotrexate and Cytosine Arabinoside on
ylase Activity. In the case of the enzyme involved in spermine Endogenous Levels of Putrescine, Spermidine, and Spermine.
synthesis, the activity level in the tumor by Day 3 is similar to In the control spleens, there appears to be an equimolar
the level in the normal spleen. However, by Day 6, the activity concentration of spermidine and spermine, and only a small
is markedly elevated and remains elevated by Day 8. When amount of putrescine is detectable (Table 3). By 3 days after
methotrexate is administered to the tumorous mice on Days 1 tumor inoculation, there is an increase in the spermidine
through 9 and the level of spermidine-stimulated 5-adenosyl- concentration and in the ratio of spermidine to spermine. This
L-methionine decarboxylase activity is measured, it is increase is more dramatic by Day 6, and there is nearly a
depressed below control levels through Day 6 and is slightly doubling of the spermidine pool by Day 8. During the entire
elevated by Day 8. This is further evidence that the polyamine course of tumor growth, the spermine concentration actually
biosynthetic pathway escapes the depressive effects of the decreases to about two-thirds of that of the original pool.
drug by Day 8. After cytosine arabinoside administration, the When the tumor-inoculated mice also receive methotrexate,
enzyme is elevated above control levels at Days 3 and 6 and is the pattern of polyamine accumulation is markedly depressed.
elevated still further by Day 8. Indeed, when either By Day 3, the spermidine pool has decreased, whereas the
methotrexate or cytosine arabinoside is administered to spermine pool remains fairly constant. Thereafter, the
healthy animals, the spermidine-dependent 5-adenosyl-L- spermidine and spermine pools drop to even lower levels. By
NOVEMBER 1972 2461
Diane H. Russell
Day 8, the spermidine pool is about one-third ofthat found in ACKNOWLEDGMENTS
the tumor-bearing mice on the same date. Cytosine arabinoside
administration also causes a marked decrease in the ability of The technical assistance of Mrs. Inez A. Hawk and Mr. Thomas A.
the tumor to accumulate polyamines, particularly spermidine. McVicker is gratefully acknowledged.
However, the spermidine pool does not decrease to the low
level found after methotrexate administration. When either of
the drugs is administered to control mice, there is also a
decrease in the polyamine pools. However, in this case, the
amounts of both spermidine and spermine are decreased, and REFERENCES
the ratio of spermidine to spermine remains similar to that
found in the controls.
1. Dykstra, W. J., Jr., and Herbst, E. J. Spermidine in Regenerating
Liver: Relation to Rapid Synthesis of Ribonucleic Acid. Science,
2. Feldman, M. J., Levy, C. C., and Russell, D. H. The Purification of
In an earlier paper (9), we reported that there is marked 5-Adenosyl-L-methionine Decarboxylase from Rat Liver: Inability
polyamine biosynthesis and accumulation in mouse L1210 s.c. to Separate Decarboxylation from Spermidine Synthesis. Biochem.
Biophys. Res. Commun., 44: 675-681, 1971.
tumors. We also reported that certain drugs cause decreased
3. Feldman, M. J., Levy, C. C., and Russell, D. H. Purification and
pools of polyamines in the tumor by Day 10 (9). These Characterization of 5-Adenosyl-L-methionine Decarboxylase from
decreases are due to decreased cellular levels of polyamines. Rat Liver. Biochemistry, //: 671-677, 1972.
In this study, data are presented that indicate that the 4. Kay, J. E., and Cooke, A. Ornithine Decarboxylase and Ribosomal
administration of either cytosine arabinoside alone or RNA Synthesis during the Stimulation of Lymphocytes by
methotrexate alone to healthy mice alters the activities of the Phytohaemagglutinin. Federation European Biochem. Soc. Letters,
polyamine-biosynthetic enzymes in the spleen. The net effect 16: 9-12, 1971.
is to decrease the ability of the spleen to synthesize and 5. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
accumulate spermidine and spermine. Methotrexate, the most Protein Measurement with the Folin Phenol Reagent. J. Biol.
Chem., 193: 265-274, 1951.
effective drug in terms of its antileukemic properties, leads to
6. Pegg, A. E., and Williams-Ashman, H. G. On the Role of
the biggest drop in the spermidine and spermine pools, both in S-Adenosyl-L-Methionine in the Biosynthesis of Spermidine by Rat
the tumor-plus-drug group and in the drug group alone. The Prostate. J. Biol. Chem., 244: 682-693, 1969.
changes in polyamine levels that occur in the drug groups 7. Raina, A., and JÃ¤nne,J., and SÃ»mes,M. Stimulation of Polyamine
alone probably reflect the extent to which the drug inhibits Synthesis in Relation to Nucleic Acids in Regenerating Rat Liver.
normal spleen cells. Therefore, the increment in synthesis due Biochim. Biophys. Acta, 123: 197-201, 1966.
to tumor invasion can be separated from those that are 8. Russell, D. H. Putrescine and Spermidine Biosynthesis in the
attributable to other factors. Development of Normal and Anucleolate Mutants of Xenopus
In the tumor group, the ratio of spermidine to spermine laevis. Proc. Nati. Acad. Sei. U. S., 68: 523-527, 1971.
increases throughout the time course of tumor growth, from a 9. Russell, D. H., and Levy, C. C. Polyamine Accumulation and
control ratio of 0.99 to a ratio of 2.1 by Day 8. In the Biosynthesis in a Mouse L1210 Leukemia. Cancer Res., 31:
methotrexate-treated tumorous mice, the ratio remains near 1 248-251, 1971.
for the 1st 8 days and, in the cytosine arabinoside-treated 10. Russell, D. H., Medina, V. J., and Snyder, S. H. Polyamine
Turnover in Normal and Regenerating Rat Liver and Brain. J. Biol.
tumorous mice, the ratio rises to and remains around 1.4 Chem., 245: 6732-6738, 1970.
through the 1st 8 days. In the groups given drugs only, the 11. Russell, D. H., and Potyraj, J. J. Spermine Synthesis in the
ratio is always near 1, indicating that in these groups there is a Castrated Rat Uterus in Response to Estradici 17 ÃŸ. Biochem. J.,
similar decrease in both the spermidine and spermine pools. A 128: 1109-1115, 1972.
high ratio of spermidine to spermine may be indicative of the 12. Russell, D. H., and Synder, S. H. Amine Synthesis in Rapidly
rapid growth process in mammalian systems. Therefore, the Growing Tissues: Ornithine Decarboxylase Activity in Regenerat
ability of a drug to lower this ratio in the tumors may be ing Rat Liver, Chick Embryo, and Various Tumors. Proc. Nati.
Acad. Sei. U. S., 60: 1420-1427, 1968.
directly proportional to the antileukemic potential of that
13. Russell, D. H., and Snyder, S. H. Amine Synthesis in Regenerating
Rat Liver: Effect of Hypophysectomy and Growth Hormone on
It is proposed that a routine assay of the amount of Ornithine Decarboxylase. Endocrinology, 84: 223-228, 1969.
spermidine and spermine present in tumor cells on Day 6 after 14. Snyder, S. H., Kreuz, D. S., Medina, V. J., and Russell, D. H.
tumor inoculation may give a rather accurate appraisal of the Polyamine Synthesis and Turnover in Rapidly Growing Tissues.
antileukemic properties of drugs given during the early course Ann. N. Y. Acad. Sci., 171: 749-771, 1970.
of the tumor development. Further work is in progress in this 15. Tabor, H., and Tabor, C. W. Spermidine, Spermine and Related
laboratory to corroborate this theory. Amines. Pharmacol. Rev., 16: 245-300, 1964.
2462 CANCER RESEARCH VOL. 32