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[CANCER RESEARCH 33, 1290 1294. June 1973]
Differential Cell Permeability and the Basis for Selective Activity of
Methotrexate during Therapy of the L1210 Leukemia1
Francis M. Sirotnak and Ruth C. Donsbach
Sloan-Kettering
Institute for Cancer Research,
New York, New York 10021
SUMMARY
The relationship of cell permeability phenomena to the
selective activity of methotrexate (MTX) during therapy of
the L1210 leukemia was examined. Drug uptake and loss in
normal tissue (small intestine) and L1210 cells in the
peritoneal cavity were measured following the administra
tion of single s.c. doses of 3, 0.3, and 0.03 mg of MTX per
kg. Within 1 hr after a dose of 3 mg/kg, drug levels in
L1210 cells and small intestine were, respectively, four- and
sixfold greater than the dihydrofolate reducÃ-asecontent.
After 0.3 mg/kg, the initial rate of uptake in both cases was
similar, but the intracellular level exceeded the enzyme
content in only L1210 cells. After a dose of 0.03 mg/kg,
uptake was almost nonexistent in small intestine but still
appreciable in L1210 cells. Free MTX was lost from small
intestine far more rapidly then from L1210 cells. After 3
mg/kg, drug level in intestine fell to enzyme level in 3 hr,
but in L1210 cells the fall required 18 to 20 hr. After a dose
of 0.3 mg/kg, approximately 8 hr were required for the drug
level in L1210 cells to reach enzyme level.
Following a dose of 3 mg of MTX per kg, administered
i.p., initial uptake was more rapid and reached higher
intracellular levels in both L1210 cells and small intestine
when compared to that which occurred following the same
dose given s.c. Drug also persists in L1210 cells in the
peritoneal cavity, but not small intestine, for a somewhat
longer period of time following i.p. administration. Uptake
and loss of MTX following a dose of 3 mg/kg s.c. is the
same in small intestine from normal and leukemic mice.
Incorporation of deoxyuridine-3H into DNA of both
small intestine and L1210 cells was almost completely in
hibited by 1 hr after a s.c. dose of 3 mg/kg. Resumption
of incorporation required 4 hr in small intestine but 14 to
16 hr in L1210 cells. The results reveal a far greater per
sistence of free MTX and a more sustained metabolic ef
fect in L1210 cells than in small intestine. This is attributed,
at least in part, to a more effective influx of drug in L1210
cells at low plasma levels.
INTRODUCTION
The basis for the selective action of effective anticancer
drugs has, for the most part, been difficult to elucidate. This
' Supported in part by Grant CA-08748 from the National Cancer
Institute.
Received January 8, 1973: accepted March 16, 1973.
1290
is particularly true with the antifolates, a class of anticancer
agents in clinical use for more than 2 decades (5). The
antifolates are excellent inhibitors of dihydrofolate reduc
Ã-asefrom both tumor cells and normal tissue (1,2, 12, 23,
32). The consequence of this inhibition for DNA synthesis
probably accounts for the ultimate cytoloxic manifeslalion
observed (17, 20). However, since both normal tissue and
drug-sensitive tumor cells contain roughly the same amounl
of Ihis "largel" enzyme (1, 2, 9, 12, 23, 22, 23, 26, 32) and
enzyme from responsive and unresponsive lumors are
inhibited essentially to the same extenl (1,2, 12, 23, 26, 32),
it is unlikely thai the interaction of drug at this sile can
account for the seleclive effecl seen during therapy. On the
other hand, only therapeutically responsive tumors have
been shown (3, 4, 68, 10, 18 20, 29 31) to possess an
efficient mechanism for transporting and concentrating
antifolates intracellularly. Although this correlation has
provocative therapeutic implications, its relevance to selec
tive activity is not at all clear, since normal cells, notably in
small intestine, liver, kidney, and hematopoietic tissue, are
also able to accumulate these drugs rapidly (19, 22, 24, 25,
27, 28).
In an effort to assess more clearly the role played by
permeability in an actual therapeutic situation, we have
made a direct examination of drug uptake in normal tissue
and tumor cells following the administration of MTX2 to
animals bearing the L1210 leukemia. Additional data on the
incorporation of UdR-3H into DNA at different intracellu
lar drug levels are also presented.
MATERIALS
AND METHODS
L1210 cells (line V) were transplanted in C57BL/6 x
DBA2 male mice as previously described (15). MTX was
provided by Lederle Laboratories, Pearl River, N. Y. Three
days prior to termination, drug was given and the LI210
cells were harvested from the peritoneal cavity of sacrificed
animals by injecting 3 ml of cold 0.14 M NaCl-0.02 M
sodium phosphate, pH 7.4, opening the cavity, and draining
the cell suspension. If the ascitic fluid was hemorrhagic,
cells were resuspended briefly in buffered 0.01 MNaCl-0.002
M sodium phosphate, pH 7.4, to lyse red blood cells. The
L1210 cells were washed twice with buffered 0.14 M
NaCl-0.02 M sodium phosphate, pH 7.4, and drug was
extracted by heating (30). After removal of the L1210 cells,
the small intestine was surgically removed and placed in
2The abbreviations used are: MTX. methotrexate; UdR-3H. deoxyuridine-3H.
CANCER RESEARCH
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Differential Cell Permeability to MTX
cold 0.14 M NaCl-0.02 M sodium phosphate, pH 7.4. The
organ was opened longitudinally, cleaned, homogenized,
and heated to extract drug in a manner described previously
(22). Small intestine from nonleukemic animals was also
removed and extracted in the same way. The drug content in
both extracts was determined by enzymatic assay (29).
Values are averages based on determinations with 2 to 4
animals. The dihydrofolate reducÃ-asecontent in unheated
extract from both normal and tumor cells was obtained by
titration with antifolate (22, 25, 32).
The incorporation of UdR-3H into DNA was determined
at varying times after the administration of MTX. For
measurements in L1210 cells, 0.5 ml of 0.75 HIMUdR-3H (2
nC\) was given i.p. in a 20- to 30-min pulse. For measure
ments in small intestine, 0.2 ml of 0.19 mM UdR-3H (2 /iCi)
was given s.c. in a 20- to 30-min pulse. The DNA was
extracted (21) and radioactivity was determined by scintilla
tion counting. Incorporation of label into DNA was linear
for at least 40 min in each case. Values given are average of
determinations on 3 to 5 animals.
RESULTS
MTX was administered s.c. to leukemic animals at 3
different doses: 3, 0.3, and 0.03 mg/kg. The lowest dose is
well below that required for minimum antileukemic activity
(13, 15). On a dosage schedule of every other day, 0.3
mg/kg would be expected to have a slight effect, whereas 3
mg/kg will achieve a maximum, or nearly maximum, an
tileukemic effect [approximately 150% increase in average
survival time (13, 15)].
The uptake of MTX by leukemia cells in the peritoneal
cavity was compared to that which occurs in the small
intestine. This comparison was made because the crypt
epithelium of the small intestine appears to be the most
drug-sensitive normal tissue in mice (22). The s.c. route of
administration was originally selected, since it would proba
bly not favor the initial uptake of MTX by L1210 cells in the
peritoneal cavity. The data obtained on uptake by these
cells, therefore, can reasonably be expected to be similar to
uptake by L1210 cells elsewhere in the animal. The
intracellular level of drug in L1210 cells and small intestine
at various times following s.c. administration to leukemic
animals is shown in Chart 1. At 0.3 and 3 mg/kg,
accumulation of drug was somewhat more rapid in small
intestine. Maximum levels of drug were attained in this
tissue in less than 1 hr, as compared to 2 hr in L1210 cells.
The maximum amount of drug accumulated after a dose of
3 mg/kg was about the same in both types of cells. When
related to drug equivalents of dihydrofolate reducÃ-ase,the
maximum level in the small intestine was 6-fold greater but
only 4-fold greater in L1210 cells. The difference in free
MTX can be attributed to a somewhat higher level of
enzyme in L1210 cells. After a dose of 0.3 mg/kg, the level
of drug accumulated exceeded the enzyme content in L1210
cells, but not in small intestine. After the smallest amount
(0.03 mg/kg) of MTX was given, the uptake of drug in
L1210 cells, compared to small intestine, was much more
rapid and reached higher levels. In L1210 cells, drug
accumulation approached enzyme level in less than 1 hr but
only 5 or 10%of enzyme level in small intestine after 2 hr.
There is a dramatic difference (Chart 3) in the rate at
which free MTX was lost from L1210 cells and intestinal
tissue. The content of drug in the small intestine was at
enzyme level within 3 hr after 3 mg/kg. By comparison,
drug content of the L1210 cells did not reach enzyme level
until 18 to 20 hr after the administration of the same dose.
Even after giving 0.3 mg/kg, drug content of L1210 cells did
not reach enzyme level until nearly 8 hr. It is also noted that
loss of drug from dihydrofolate reducÃ-asein both L1210
cells and small intestine occurred to a significant extenl,
although slowly, within ihe period of the experiment. This
result is in agreement wilh prior observalions made on the
rate of drug loss from dihydrofolate reducÃ-asein small
intestine (25).
The level of uptake of MTX by L1210 cells and small
intesline was also examined after ihe i.p. injeclion of drug.
This route of injection results in only a slight, if any,
increase in therapeutic effect when compared to the s.c.
route (D. J. Hulchison, private communication). However,
a more rapid inilial uptake of drug by L1210 cells in the
peritoneal cavity would be expected. A comparison of ihe
results obtained after 3 mg/kg were administered by each
route is shown in Chart 2. As anticipated, MTX was concenlraled in L1210 cells lo much higher levels following i.p.
injeclion. The intracellular level in 1 hr was 15times grealer
lhan ihe dihydrofolale reducÃ-asecontenÃ-.The accumulasmall intestine
lleukemic mice)
small intestine
lleukemic mice)
J.Omg/Kg 1
0.3mg/kg U.c.
- 3.0mg/kg 1
0.03 mg/kg J
: 0.03mg/kg J
•¿
0.3mg/kg
i 3mg/kg s.c.
> 3mg/kg i.p.
L s.c
dinydrofolate reducÃ-ase
dihydrofolate reducÃ-ase
16
t(hours!
8
12
16
t(hours!
Chart I. The uptake and loss of MTX in L1210 cells and small intestine
after s.c. administration of different amounts to leukemic mice.
0
5
10
IIhours)
15
20
"0
5
10
15
X
I(hours!
Chart 2. The uptake and loss of MTX in L1210 cells and small intestine
after s.c. or i.p. administration of 3 mg/kg to leukemic mice.
JUNE 1973
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1291
Francis M. Sirotnak
and Ruth C. Dons bach
tion in LI2IO cells, following the same dose given s.c., is
also shown for comparison. Although very high levels were
intestine{leukemic
small
achieved by the i.p. route, they were found to diminish
mice)1
rapidly. Even so, after 3 mg/kg, the intracellular level was
s.c.1\
3 mg/kg
still about twice the enzyme content in 20 hr.
The initial uptake of MTX by small intestine was also
reducÃ-asei
dihydrofolate
higher following an i.p. dose, although not to the same
extent seen in L1210 cells. After 3 mg/kg, drug was
i16
'4
i
2t
8
12i
concentrated to a level about 30% higher than that attained
by the s.c. route. Despite the difference in the initial uptake
of MTX, intracellular levels were down to enzyme level in
about the same time.
The greater persistence of drug in the L1210 cells in the
peritoneal cavity could explain the better therapeutic effect
of an i.p. dose. Although the survival of mice bearing this
leukemia is probably related to the pathological changes
that occur during the progression of the leukemia, the
16
20
12
8
degree of L1210 cell proliferation in the peritoneal cavity
t (hours)
might also be of some importance.
Chart 4. Intracellular MTX levels and the incorporation of UdR-3H
In a related experiment, the uptake and subsequent loss of into DNA of small intestine at varying intervals following the administra
MTX in small intestine was also studied in nonleukemic tion of 3 mg/kg to leukemic mice. The level of radioactivity incorporated
animals. A comparison with "leukemic" intestine was of in controls was 6000 to 8000 cpm/g of intestine.
special interest, since it has been previously shown (14) that
a marked diminution in the size of the small intestine occurs
during the development of the leukemia. One might also
L1210cells
anticipate a corresponding change in metabolic disposition
3mg/kgs.c.
which could alter drug distribution in this tissue. The results
obtained after a dose of 3 mg/kg was given is shown in
Chart 3. The rate and amount of drug uptake and the loss of
drug with time was quite similar in small intestine from
normal and leukemic animals. The content of dihydrofolate
reducÃ-asewas also found to be about the same in both
tissues.
A direct indication of the metabolic consequences of
various intracellular drug levels was obtained by measuring
UdR-3H incorporation into DNA after the administration
of 3 mg of MTX per kg. The data on the relative extent of
UdR-3H incorporation are given in Charts 4 and 5. The
values given represent the percentage of incorporation
obtained at each interval when compared to the amount of
12
20
8
16
incorporation obtained in a group of untreated controls.
t (hours)
SJXtm^ten108642nTAI
Chart 5. Intracellular MTX levels and the incorporation of UdR-3H
into DNA of L12IO cells at varying intervals following the administration
of 3 mg/kg to leukemic mice. The level of radioactivity incorporated in
controls was 3 to 4 x 10s cpm/g of cells.
10.-
small intestine
3mg/kg s.c.
leukemic mice
normal mice
Chart 3. The uptake and loss of MTX in small intestine from normal
and leukemic mice after s.c. administration of 3 mg/kg.
1292
The level of MTX in L1210 cells and small intestine at
various intervals is also shown. Cessation of UdR-3H
incorporation into the DNA of both L1210 cells and
intestine is nearly complete in 1 hr. This is in agreement
with the amount of drug uptake obtained in both cases.
Resumption of UdR-3H incorporation occurred within 4 hr
in small intestine (Chart 4). At this point, the level of MTX
was already below enzyme level. Recovery of UdR-3H
incorporation continues and approaches maximum in 12 hr.
Further recovery after this point occurred more slowly. This
seems commensurate with the very gradual loss of drug
from enzyme also observed. These results on UdR-3H
incorporation into DNA of small intestine of leukemic
animals at different drug levels are very similar to that
reported for small intestine in normal animals (20, 23).
CANCER RESEARCH
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Differential
Resumption of incorporation of UdR-3H into the DNA
of L1210 cells occurred much later then in small intestine.
No incorporation was detectable before 14 hr after the
administration of 3 mg/kg (Chart 5). However, recovery
occurred steadily thereafter, so that incorporation was at
40% of the maximum rate at 20 hr. Some discrepancy was
observed in L1210 cells relating to the degree of UdR-3H
incorporation at low-intracellular drug levels. Appreciable
incorporation of label occurred when the drug level was still
above enzyme content. This is most likely due to the partial
stoichiometry of the dihydrofolate reductase-MTX interac
tion. Inhibition of L1210 enzyme activity in vitro has been
shown to have a titration relationship to drug concentration
only to about 80% of the total number of drug binding sites
(11, 16). Also, complete inhibition of UdR-3H incorpora
tion following uptake by L1210 cells in vitro does not occur
until the intracellular drug level is nearly twice the enzyme
level (F. M. Sirotnak and R. C. Donsbach, unpublished
results).
DISCUSSION
The data clearly reveal a far greater persistence of MTX
in L1210 cells when compared to small intestine. Moreover,
the degree of persistence in L1210 cells at different dosage
correlates quite well with the antileukemic effect obtained
(Refs. 13 and 15; D. J. Hutchison, private communication).
In understanding the reason for this difference in drug loss
between the leukemia cells and normal tissue, the data
obtained on the uptake of MTX after the administration of
0.30 mg/kg are important. At this low concentration, the
rate of uptake within the 1st 0.5 hr in L1210 cells is about 1.0
times higher than that seen in small intestine. Since levels of
MTX in plasma fall very rapidly soon after administration
(22, 24, 25), this greater ability of L1210 cells to accumulate
drug at low concentration becomes a critical factor in
maintaining intracellular levels above the dihydrofolate
reducÃ-asecontent.
Assuming only minimum differences related to drug
distribution within body fluids, the approximate rate of drug
uptake by L1210 cells and small intestine obtained within
the 1st hr after the administration of varying amounts would
suggest a mechanism for concentrating drug in each case,
which is about equal in capacity but more efficient in L1210
cells. A greater affinity of MTX for the mechanism in
L1210 cells could account for a more efficient operation at
low-plasma concentrations. Studies on uptake by L1210
cells in vitro provide (7, 18, 29-31) ample evidence for an
exceptionally high affinity of the carrier-transport system
for MTX.
Overall, the results provide evidence for a basis for
selective activity of MTX in the L1210 cell system, which is
at least partially related to differences in cell permeability
between target tumor cells and normal host tissue. They do
not, by themselves, exclude the possibility that other
physiological differences may exist as well not only in this
system but in other host-tumor systems and patients with
responsive leukemias.
JUNE
Cell Permeability
to M TX
ACKNOWLEDGMENTS
We acknowledge with gratitude the interest and advice of Drs. Dorris J.
Hutchison and Frederick S. Philips during the course of these studies. We
are also grateful to Dr. Franz A. Schmid for assistance in providing
leukemic mice.
REFERENCES
1. Baker, B. L. Design of Active Site-Directed Irreversible Enzyme
Inhibitors, pp. 192 263. New York: John Wiley and Sons, Inc., 1967.
2. Blakley, R. L. The Biochemistry of Folie Acid and Related Pteridines.
pp. 139-181. New York: John Wiley and Sons, Inc.. 1969.
3. Divekar, A. Y., Vardya, N. R.. and Braganca, B. M. Active Transport
of Aminopterin in Yoshida Sarcoma Cells. Biochim. Biophys. Acta,
135: 927-936, 1967.
4. Divekar, A. Y., Vardya, N. R.. and Braganca. B. M. Defective
Transport of Aminopterin in Relation to the Development of Resist
ance in Yoshida Sarcoma Cells. Biochim. Biophys. Acta, 135:
937-946, 1967.
5. Farber, S., Diamond, L. K., Mercer. R. D., Sylvester, R. F., Jr., and
Wolff. J. A. Temporary Remissions in Acute Leukemia in Children
Produced by Folie Acid Antagonists, 4-Aminopteroyl-glutamic Acid
(Aminopterin). New Engl. J. Med., 238: 787 793, 1948.
6. Fisher. G. A. Defective Transport of Amethopterin (Methotrexate) as
a Mechanism of Resistance to the Antifolate in L5I78Y Leukemia
Cells. Biochem. Pharmacol., //: 1233-1234, 1960.
7. Goldman, I. D., Lichenstein, N. S., and Oliverio, V. T. Carriermediated Transport of the Folie Acid Analogue, Methotrexate. in the
L1210 Leukemia Cell. J. Biol. Chem., 243: 5007 5017, 1968.
8. Hakala, M. T. On the Nature of the Permeability of Sarcoma-180
Cells to Amethopterin in vitro. Biochim. Biophys. Acta, 102: 210 225,
1965.
9. Hall, T. C., Roberts, D., and Kessel, D. H. Methotrexate and Folie
ReducÃ-asein Human Cancer. 2: 135 142, 1966.
10. Harrap, K. R., Hill, B. T.. Furness. M. E.. and Hart, L. I. Sites of
Action of Amethopterin: Intrinsic and Acquired Drug Resistance.
Ann. N. Y. Acad. Sci., 186: 312 324, 1971.
11. Hillcoat, B. L., Perkins, J. P.. and Berlino. J. R. Dihydrofolate
ReducÃ-asefrom ihe L1210R Murine Lymphoma. Further Studies on
the Binding of Substrates and Inhibitors to the Enzyme. J. Biol.
Chem., 242:4777 4781, 1967.
12. Huennekens, F. M., Dunlop, R. B., Freisheim, J. H., Gunderson, L.
E., Harding, N. G. L., Levison, S. A., and Meli, G. P. Dihydrofolate
ReducÃ-ase:Structural and Mechanistic Aspects. Ann. N. Y. Acad.
Sci., 186: 85 99, 1971.
13. Hutchinson. D. J. Quinazoline Anlifolates: Biologic Activilies. Cancer
Chemotherapy Rept, 52: 697 705, 1968.
14. Hutchison, D. J., Philips, F. S., Schmid, F. A., Sodergren, J. E., and
Sternberg, S. S. Inhibilion of Hosl Proliferative Tissue by LI2IO
Leukemia: The Inverted Effect of Metholrexale (MTX). Proc. Am.
Assoc. Cancer Res., 13: 18, 1972.
15. Hulchison, D. J., Robinson, D. L., Marlin, D., Ittensohn, O. L., and
Dillenberg. J. Effects of Selected Anticancer Drugs on the Survival
Time of Mice with L12IO Leukemia: Relalive Responses of Anlimetabolile-resislant Strains. Cancer Res.. 22: 57 72, 1962.
16. Hutchison, D. J., Sirotnak, F. M., and Albrecht, A. M. Dihydrofolate
ReducÃ-ase Inhibilion by the 2.4-Diaminoquina/oline Anlifolates.
Proc. Am. Assoc. Cancer Res., 10: 41, 1969.
17. Karnofsky, D. A., and Clarkson, B. D. Cellular Effects of Anticancer
Drugs. Ann. Rev. Pharmacol.. 3: 357 428. 1963.
18. Kessel, D., and Hall, T. C. Amethoplerin Transpon in Ehrlich Asciles
Carcinoma and L12IO Cells. Cancer Res.. 27: 1539 1543, 1967.
19. Kessel, D., Hall. T. C., and Roberts, D. Modes of Uplake of
1973
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
1293
Francis M. Sirotnak and Ruth C. Dons bach
20.
21.
22.
23.
24.
25.
Melholrexale by Normal and Leukemic Human Leukocytes in Vitro
and Their Relationship to Drug Response. Cancer Res., 28: 564 570,
1968.
Kessel, D., Hall, T. C„Roberts, D., and Wodinsky, I. Uptake as a
Determinant of Methotrexate Response in Mouse Leukemias.
Science, 150: 752-754, 1965.
Lenaz, L., Sternberg, S. S., and Philips, F. S. Cytotoxic Effects of
l-/}-D-Arabinofuranosyl-5-fluorocylosine and l-/3-n-Arabinofuranosyl-cytosine in Proliferating Tissue in Mice. Cancer Res., 29:
1790 1798. 1969.
Margolis, S., Philips, F. S., and Sternberg, S. S. The Cytotoxicity of
Methotrexate in Mouse Small Intestine in Relation to the Inhibition of
Folie Acid ReducÃ-aseand of DNA Synthesis. Cancer Res.. 31:
2037 2046, 1971.
McCullough, J. L., and Berlino, J. R. Dihydrofolate ReducÃ-asefrom
Mouse Liver and Spleen, Purification, Properties and Inhibition by
Substituted 2,4-Diaminopyrimidines and 4,6-diaminotriazines. Biochem. Pharmaco!.. 20: 561 574, 1971.
Oliverio. V. T.. and Zaharko, D. S. Tissue Distribulion of Folale
Anlagonists. Ann. N. Y. Acad. Sci.. 186: 387 399. 1971.
Philips, F. S., Sirotnak. F. M.. Sodergren. J. E., and Hutchison, D. J.
Uplake of Metholrexate, Aminopterin, and Melhasquin and Inhibi-
1294
26.
27.
28.
29.
30.
31.
32.
lion of Dihydrofolale ReducÃ-aseand of DNA Synlhesis in Mouse
Small Intestine. Cancer Res., 33: 153-158, 1973.
Roberts, D., Wodinsky, I., and Hall. T. C. Studies of Folie ReducÃ-ase.
III. The Level of Enzyme Aclivity and Response lo Melholrexale of
Transplanlable Mouse Tumors. Cancer Res., 25: 1899 1903, 1965.
Rubin, R. C.. Henderson, E. S. Owens. E. S., and Rail, D. Uptake of
Metholrexate-3H by Rabbil Kidney Slices. Cancer Res., 27: 553-557,
1967.
Rubin, R. C., Owens, E., and Rail, D. Transpon of Methotrexate by
Choroid Plexus. Cancer Res., 28: 689 694, 1968.
Sirotnak, F. M., and Donsbach. R. C. Comparative Studies on the
Transport of Aminopterin, Melholrexate, and Methasquin by ine
LI210 Leukemia Cell. Cancer Res., 32: 2120 2126, 1972.
Sirolnak, F. M., Kurita. S., and Hutchison. D. J. On the Nature of a
Transport Alteralion Delermining Resislance lo Amelhoplerin in ihe
L1210 Leukemia. Cancer Res.. 28: 75-80, 1968.
Sirolnak, F. M., Kurila, S., Sargenl, M. G., Robinson. D. L.. and
Hulchison, D. J. Sequenlial Biochemical Alleralion to Antifolate
Resistance in LI210 Leukemia. Nature 216: 1236-1237. 1967.
Werkheiser. W. C. The Biochemical, Cellular, and Pharmacological
Action and Effects of the Folie Acid Aniagonisls. Cancer Res., 23:
1277-1285, 1963.
CANCER RESEARCH
VOL. 33
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Differential Cell Permeability and the Basis for Selective Activity
of Methotrexate during Therapy of the L1210 Leukemia
Francis M. Sirotnak and Ruth C. Donsbach
Cancer Res 1973;33:1290-1294.
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