(CANCER RESEARCH 32, 390-397,
February 1972)
Role of Catabolism in Pyrimidine Utilization for Nucleic Acid
Synthesis in Vivol
Geoffrey M. Cooper,2 W. F. Dunning, and Sheldon Greer
Departments of Biochemistry (G. M. C., S. G./, Medicine [W. F. £>./,and Microbiology ¡S.G./, University of Miami, Coral Gables, Florida 33146,
and Papanicolaou Cancer Research Institute fW. F. D./, Miami, Florida 33136
SUMMARY
is an irreversible inhibitor of dihydrouracil dehydrogenase, the
first and rate-limiting enzyme in the catabolism of the
The incorporation of pyrimidines into nucleic acids in vivo pyrimidine bases (4). The results of kinetic studies with a
is increased by inhibition of pyrimidine catabolism with crude enzyme preparation from rat liver were consistent with
diazouracil. The utilization of iodouracil or thymine for DNA the possibility that the mechanism of irreversible inhibition by
synthesis
can be increased approximately
20-fold by DU involves covalent bond formation at the enzymic active
simultaneous administration
of diazouracil and a purine site (10). This report deals with the effect of DU on the
deoxyribonucleoside.
The incorporation of iodouracil and utilization of pyrimidine bases and nucleosides for nucleic acid
thymine, when administered at high doses, is elevated to synthesis in vivo.
nearly that obtained with the corresponding pyrimidine
In Ehrlich ascites cells (17) and human leukocytes (2), the
deoxyribonucleosides, while at low doses thymidine is utilized incorporation of thymine or halogenated base analogs into
preferentially
over thymine.
Diazouracil
and purine DNA appears to be limited largely by the availability of
synthesis.
deoxyribonucleosides
do not
appreciably
affect
the deoxyribose 1-phosphate for deoxyribonucleoside
incorporation of thymidine or iododeoxyuridine into DNA. The synthesis of ribonucleosides (5, 36) and ribonucleotides
The utilization of fluorouracil and uracil is also elevated by (37) from uracil and FU is also limited by the availability of
in
diazouracil but is not significantly affected by purine ribose 1-phosphate and phosphoribosyl pyrophosphate
ribonucleosides. Diazouracil has a similar effect on pyrimidine some, but not all, murine tumors which have been studied (17,
incorporation in cells of the Dunning leukemia, rat liver, 24). In addition to limitation by the availability of reactants
spleen, and small intestine, in spite of the differences in for anabolic conversion, the utilization of free pyrimidines,
catabolic activity between these tissues, a finding that particularly in vivo, might also be limited by their catabolism.
indicates the importance of systemic catabolism. The toxic The present study indicates that this is the case, since
and antitumor activities of fluorouracil are potentiated equally inhibition of catabolism by administration of DU enhances the
by diazouracil administration.
incorporation of pyrimidine bases into nucleic acids.
A preliminary report of some of this work has been
presented (8).
INTRODUCTION
Pyrimidine
catabolism
may
be
relevant
to the
chemotherapeutic activity of the fluorinated pyrimidines (7,
19, 20) and to the possible therapeutic use of the brominated
and iodinated pyrimidines as tumor radiosensitizing agents (1,
11, 18, 26, 43). One approach to elucidating the role of
catabolism is the development of inhibitors of enzymes of the
catabolic pathway. Such inhibitors may also be of therapeutic
interest in the potentiation of the antineoplastic activity of the
halogenated pyrimidines.
Previous work in our laboratory (9, 10) indicated that DU3
'This investigation was supported by Grant DRG-1076 from the
Damon Runyon Memorial Foundation, and by Grant ÇAI2522 and
Contract PH 43-64-80 from the National Cancer Institute, NIH.
"Predoctorat trainee supported by USPHS Training Grant HE-05463
from the National Heart and Lung Institute and a Robert E. Maytag
Fellowship from the University of Miami.
3The abbreviations used are: DU, 5-diazouracil; FU, 5-fluorouracil;
IU, 5-iodouracil; lUdR, 5-iododeoxyuridine.
Received August 12. 1971; accepted November 3, 1971
390
MATERIALS AND METHODS
Chemicals. 125IU, 12SIUdR, and FU-6-3H were purchased
from Amersham/Searle
Corporation,
Des Plaines, 111.
Uracil-6-3H, thymine-methyl-3H,
and thymidine-methyl-3H
were purchased from New England Nuclear, Boston, Mass. DU
was purchased from Sigma Chemical Company, St. Louis, Mo.
Unlabeled pyrimidines, pyrimidine nucleosides, and purine
nucleosides were purchased from either Sigma Chemical
Company or Calbiochem, Los Angeles, Calif. Actinomycin D is
a product of Merck Sharp and Dohme, West Point, Pa.
Tumor-bearing Animals. The Dunning leukemia IRC 741
(13) was maintained by unilateral s.c. transplantation in the
flank of adult male Fischer 344 rats. Animals were either bred
by W. F. Dunning or purchased from Microbiological
Associates, Bethesda, Md., or from Charles River Breeding
Laboratory, Wilmington, Mass. Purina laboratory chow and
water were provided ad libitum.
Administration of Radioactive Pyrimidines. DNA precursors
(I2SIU,
125IUdR, 3H-labeled thymine, and 3H-labeled
CANCER RESEARCH VOL. 32
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In Vivo Inhibition of PyrÃ-midine CataboUsm
thymidine) were administered daily for a 4-day period collected either by centrifugation or winding on a glass rod.
beginning when the transplanted tumors were first palpable, The precipitate was washed 3 times with 95% ethanol and
usually 9 or 10 days after implantation. 3H-Labeled FU and dissolved in 5 ml of 15 mM NaCl plus 1.5 mM sodium citrate.
3H-labeled uracil were administered in a single injection when
3H was determined by liquid scintillation counting.
the tumors had grown to a diameter of approximately 3 cm,
generally about
14 days after implantation.
DU and
methotrexate were injected 2 hr prior to administration of the
radioactive pyrimidines. Furine nucleosides were injected
immediately after pyrimidine administration. All chemicals
were administered i.p. in 0.85% NaCl solution. The animals
were sacrificed 24 hr after injection, and tissues to be studied
were removed and stored at —¿70°.
Extraction of Nucleic Acids. For extraction of 3H-labeled
•¿[T
nucleic acids, approximately 3 g of tissue were homogenized in
a Sorvall Omnimixer in 10 ml of distilled water at 0 . Fifteen
ml of 10% perchloric acid were added, and the precipitate was
collected by centrifugation, washed twice with 30 ml of 5%
perchloric acid, and extracted 3 times with 30 ml of
ethanol : ether (1:1). Nucleic acids were then hydrolyzed by
heating at 90° for 45 min in 15 ml of 5% perchloric acid.
4SSuS
Insoluble material was removed by centrifugation.
125I-Labeled DNA was extracted by a modification of the
2!E
method of Marmur (27). Approximately 2 g of tissue were
homogenized in 10 ml of 150 mM NaCl plus 15 mM sodium
citrate at 0°in a Sorvall Omnimixer. Sodium dodecyl sulfate
-
«"I
1I 8
7IPI
6•»MMif
33
1-f-rn-
Contini
2 E 12 24 4l
Tile after DU administration{t[|
was added to a final concentration of 4%, and the material was
Chart 2. Recovery of dihydrouracil dehydrogenase activity after DU
further homogenized in a glass tissue grinder, followed by administration. At varying times after DU administration (5 mg/kg),
heating at 60°for 30 min. The preparation was then cooled to animals (Sprague-Dawley females) were sacrificed for determination of
room temperature, NaCl was added to a final concentration of liver dihydrouracil dehydrogenase specific activity. Control animals did
l M, and insoluble material was removed by centrifugation at not receive DU. Data from a representative experiment are presented as
0°for 30 min at 13,000 X g. DNA was precipitated from the in Chart 1.
supernatant fluid by addition of 2 volumes of 95% ethanol and
'= 12
Z
6
Ì4
5
S
i
2
-
2 24
•¿
f
2 24
rrt
2 24
lime il Sacrilice tir
Nue
ti
DU
DO»e
Administered
Chemicals
1
2
u usi
4
Chart 1. Inhibition of dihydrouracil dehydrogenase in vivo by DU.
Two hr after i.p. injection of DU, animals were sacrificed and the
specific activity of their liver dihydrouracil dehydrogenase was
determined. Sprague-Dawley females were used. Data are represented as
mean ±S.D. of duplicate animals from a representative experiment.
Chart 3. Inhibition by actinomycin D of recovery of dihydrouracil
dehydrogenase activity following DU administration.
DU was
administered as a single injection of 5 mg/kg. Actinomycin D (AD), l
mg/kg, was administered at the same time as DU and again 12 hr later.
Animals (Sprague-Dawley females) receiving DU and/or actinomycin D
were sacrificed at 2 and 24 hr after initial injection for determination of
liver dihydrouracil dehydrogenase specific activity. Data are represented
as mean ±S.D. of 2 to 8 animals. In animals receiving DU alone, the
activity of dihydrouracil dehydrogenase is significantly different
(p < 0.05) at 2 and 24 hr after injection, while in animals receiving DU
and actinomycin D, enzyme activity is not significantly different
(p > 0.5) at the 2 times.
FEBRUARY 1972
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391
Geoffrey M. Cooper, W. F. Dunning, and Sheldon Gréer
Quenching was corrected by the method of channel ratios.
1251 was counted in a well-type y scintillation spectrometer.
DNA and deoxyribonucleotides
were determined by the
diphenylamine
reaction. Total nucleic acid content of
hydrolysates was estimated by absorbance at 260 nm.
Enzyme Assays. Dihydrouracil dehydrogenase was assayed
in the 105,000 X g supernatant fluid of all tissues by
measuring the TPNH-dependent dehalogenation of I2SIU as
previously described (10). The reaction mixture contained 0.2
mM 12SIU (0.1 juCi), 2 mM TPNH, and 10 mM phosphate
In
Vivo Metabolism
of 12SIUdR.
Swiss mice (Karwood
Farms, New City, N.Y.) received a single i.p. injection of
12SIUdR in 0.85% NaCl solution. The mice were placed in
metabolism cages, and urine, which contained 50 to 60% of
the administered radioactivity, was collected 20 hr after
injection. A 0.5-ml urine sample was placed on a 1-ml anión
exchange column (Bio-Rad AG-1-X8-C1anion-exchange resin).
12S1U and ' 2SIUdR were eluted as a single peak with 0.01 N
HC1 and 12SI" was eluted with 5 M KI. All urinary
radioactivity chromatographed as III and lUdR or as I".
buffer, pH 7.4. The conditions for all assays were such that
product (12SI~) formation was linear with incubation time,
and enzyme activity was proportional to the amount of
enzyme extract added to the incubation mixture. Protein was
estimated by the method of Warburg and Christian (44).
Table 1
Incorporation of '3 5W and '2 sWdR into DNA
of Leukemia IRC 741
Chemicals were administered daily for a 4-day period starting 10
days after IRC 741 implantation: methotrexate, 0.1 mg/kg; DU, 5.0
mg/kg; guanosine and deoxyguanosine, 100 mg/kg; '2 5IU, 25 mg (104
Mmoles)/kg, 0.1 /uCi/Mmole; 125IUdR, 25 mg (71 Mmoles)/kg, 0.1
/iCi/jimole. Twenty-four hr after the final injection the animals were
sacrificed, and the specific activity of the isolated tumor DNA was
determined. Data are presented as nmoles of administered precursor
incorporated per mg of DNA; mean ±S.D. of the number of animals
given in parentheses. Statistical analysis of selected illustrative data was
by means of the Student t test.
Chemicals administered in
addition to methotrexate
Radioactive
DNA precursor
NoneDeoxyguanosineGuanosineDUDU
25lUdR0.11
In Vivo Inhibition of Pyrimidine Catabolism by DU. The
effect of in vivo administration of DU was studied by
determination of dihydrouracil dehydrogenase activity in the
105,000 X g supernatant
fraction of livers excised from
animals that had previously been given injections of DU.
Maximal inhibition, which varied from 80 to 95% (Charts 1 to
3), was attained with a DU dose between 2.5 and 5.0 mg/kg
(Chart 1). Similar results were obtained with both Fischer and
Sprague-Dawley rats.
Inhibition of catabolism in vivo was also determined in
studies of the metabolism of '2 5lUdR after its administration
to Swiss mice. Radioactive metabolites excreted in the urine
were analyzed 20 hr after administration of 25 mg/kg
125IUdR (1 /jCi/mouse). In duplicate mice that received 5
mg/kg of DU 2 hr before '2 5lUdR injection, 15% of the total
urinary radioactivity was 125I", whereas 12SI" constituted
sIU(nmoles/mgDNA)
53% of the radioactivity excreted by control mice not
receiving DU. Since DU does not inhibit the phosphorolysis of
0.03(8)0.520.150.621.80.752.10.19
lUdR by either thymidine
phosphorylase
or uridine
(4)a0.05
phosphorylase
(G.
M.
Cooper
and
S.
Gréer,unpublished
(2)0.10(4)°0.6 observations), the 72% inhibition of 12SJ~ formation in vivo
(9)a0.05
(2)0.3
deoxyguanosineDU
and
guanosineNoneSIU5IU5IU5IUS1U5IUMUdRDU
and
and deoxyguanosine
RESULTS
(7)1.7
0.7(10)
' Significantly different from control values (p < 0.05).
Incorporation ofl2SIU
resulting from DU administration represents inhibition of
dihydrouracil dehydrogenase.
The duration of inhibition was studied by assaying 12SIU
catabolism in extracts of livers removed from animals
sacrificed at varying times after administration of a single dose
Table 2
and '2 5WdR into DNA of liver, spleen, and small intestine
The experimental details were as described in Table 1. DNA was extracted from the indicated normal tissues of
tumor-bearing rats. Data aie represented as nmoles of administered precursor incorporated per mg DNA; mean ±S.D. of the
number of animals indicated in parentheses. Statistical analysis of selected data was by means of the Student t test.
125IU (nmoles/mg DNA)
Chemicals administered in
Radioactive
addition
precursorNoneDeoxyguanosineGuanosineDUDU
to methotrexate
DNA
intestine0.047
(4)0.050
5 ±0.005
(4)0.043
±0.002
(2)0.022
±0.010
(2)0.01±0.02
(2)0.045
0±0.001
(2)0.050
±0.002
(2)"0.21±0.010
(2)°0.23
±0.005
±0.04(4)6N.D.C0.20
(4)60.085
±0.04
deoxyguanosineDU
and
(2)0.20±0.015
guanosineNone5IU5IU5IU5IUSIU5IUMUdRSpleen0.01
and
±0.02 (2)Liver0.022
±0.05 (2)Small
(4)0.35±0.005
(2)0.065
±0.05
(2)0.20±0.005
±0.02(2)"0.88
±0.18(4)b0.20
(2)0.75±0.02
±0.05(2)
0 Significantly different from control values (p < 0.05).
6 Significantly different from control values (p < 0.01).
c N.D., not determined.
392
CANCER RESEARCH VOL. 32
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In Vivo Inhibition of Pyrimidine Catabolism
of DU (Chart 2). Dihydrouracil dehydrogenase activity doses of methotrexate inhibit tumor growth and reduce the
increased slowly and did not reach the control level until 24 to incorporation of *2 s lUdR into DNA, perhaps as a result of
reutilization of thymidine released from killed cells (3, 14, 28,
48 hr after DU administration.
The recovery of dihydrouracil dehydrogenase activity is 38). '2 s IU is incorporated to only 5% the extent of '2 s lUdR.
prevented by administration of actinomycin D (Chart 3). This Administration of deoxyguanosine, which presumably acts as a
of deoxyribose
1-phosphate,
increases
125IU
is consistent with the possibility that enzyme synthesis is source
required for recovery, as would be expected from the incorporation 5-fold. Guanosine has no effect, a fact indicating
irreversibility of DU inhibition. The effect of actinomycin D specificity for the deoxyribose moiety. Deoxyadenosine can
alone probably results from inhibition of normal enzyme substitute for deoxyguanosine.
Inhibition of pyrimidine
catabolism by DU results in a 6-fold increase in the
turnover.
Utilization of Pyrimidines for Nucleic Acid Synthesis. The incorporation of '2 5IU. Combined administration of DU and
18-fold to
effect of DU and purine nucleosides on the incorporation of deoxyguanosine increases 125IU incorporation
125IU and 12SIUdR into DNA of the IRC 741 leukemia is approximately the level attained by the administration of
illustrated in Table 1. In this experiment, methotrexate, 0.1 125IUdR (note that the molar quantity of 12SIUdR
is 30% less than that of 125IU). The
mg/kg, was administered to all animals. This is an optimal dose administered
incorporation of the deoxyribonucleoside,
125IUdR, is not
resulting in a 50 to 100% increase in 125IUdR incorporation
over that in control animals receiving I25IUdR alone. Larger significantly affected by DU and deoxyguanosine.
Table 3
Incorporation of 3H-labeled thymine and thymidine into DNA
ofleukemic tissue
Chemicals were administered daily for a 4-day period starting 9 days after IRC 741
implantation: DU, 5 mg/kg; 3H-labeled thymine and thymidine, either 10 Mmoles/kg (20
iiCi/Aimole) or 100 Mrnoles/kg (2 ¿iCi/Mmole);
deoxyguanosine, 100 mg/kg. Methotrexate was
not administered. Data are represented as nmoles of administered precursor incorporated per
mg DNA; mean ±S.D. of the number of animals given in parentheses. Statistical analysis of
selected data was by means of the Student t test.
3H-Labeled thymine (nmoles/mg DNA)
Dose of labeled precursor
Administered
chemicalsNone
DNA
precursorThymine
DU
Thymine
Deoxyguanosine
Thymine
DU + deoxyguanosine
Thymine
None
Thymidine
DU + deoxyguanosine3H-Labeled
Thymidine10
jumoles/kg0.093
±0.5 (2)
(3)
7.5 ±0.1 (2)
0.3
0.1 (2)
5.1 ±0.1 (2)
0.1 (2)
0.2
0.1 (2)°
15 ±1 (2)°
0.7
27 ±3 (2)
8.5
1.5 (2)lOO/jmoles/kg3.8
38 ±6(2)
9.0 (1)0.009
" Significantly different from control values (p < 0.01).
Table 4
Utilization of 3H-labeled FU for RNA synthesis in various tissues
Single injections of chemicals were administered 24 hr before the animals were sacrificed: 3H-labeled
FU, 20 mg (155 Mmoles)/kg (1.9 pCi//umole); DU, 5 mg/kg; deoxyguanosine and guanosine, 100 mg/kg.
Total nucleic acids were extracted and incorporation of 3H-labeled FU is expressed as dpm/A26 „¿
unit of
the nucleic acid hydrolysate. Data are represented as mean ±S.D. of the number of animals given in
parentheses. Statistical analysis of selected data was by means of the Student / test.
3H-Labeled FU (dpm/A^,,
unit)
Administered
chemicalsNoneGuanosineDeoxyguanosineDUDU
74188
(2)110±
±20
(2)72(1)340
14
guanosineDU
and
deoxyguanosinePooled
and
data:Without
DUWith
DUIRC
(2)°420
±15
(2)420
±45
(2)93
±100
intestine38
(2)21
±1
(2)20(1)62
±5
(2)°94
±2
12(2)118+2
±
(2)22
(2)34
±5
(2)42
±5
(2)93
±14
(2)°138
±9
18(2)158
±
(2)35
±34
(2)46
±6
(2)48
±6
(2)90
±4
(2)°110
±2
10(2)130±
±
30(2)44
(6)110
±7
(5)390
±21
(5)92
±3
(6)130±
±11
±120 (6)°Spleen23±29 (6)°Liver28 35 (6)"Small
±25(6)°
1Significantly different from animals not receiving DU (p < 0.05).
FEBRUARY 1972
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393
Geoffrey M. Cooper, W. F. Dunning, and Sheldon Gréer
Table 5
Comparison between levels of pyrimidine catabolism and stimulation of
pyrimidine utilization by DU in different tissues
Incorporation of 'J 5IU into DNA and 3H-labeled FU into RNA in the presence and absence of DU in different tissues was
determined as described in Tables 1 to 4. The effect of DU on FU incorporation is represented by the ratio of 3H-labeled FU
incorporated following DU administration to 3H-labeled FU incorporated without DU administration (3H dpm/A260 unit
with DU: 3H dpm/A,60 unit without DU). A similar ratio is used to represent the effect of DU on 125IU incorporation
(nmoles ' *5 lU/mg DNA with DU: nmoles '2 5lU/mg DNA without DU). The specific activity of dihydrouracil dehydrogenase
was determined in the 105,000 X g supernatant fluid of all tissues. Data are expressed as mean ±S.D. of the number of
determinations given in parentheses.
Ratio of pyrimidine incorporation with and
without administered DU°
Tissue
IRC
741SpleenSmall
intestineIntestinal
mucosaLiver<
Dihydrouracil dehydrogenase
(nmoles '2 51"/hr/mg protein)
3H-Labeled FU incorporation
(with DU:without DU)
12s IU incorporation
(withDU:withoutDU)
(5)4.4*
±0.9
(5)2.5 1.3
(6)N.D.b3.7
±0.4
(10)3.8
±0.9
(6)3.3
±0.8
(6)N.D.3.7
±0.6
(2)<
0.05
0.2
(4)2.2
(5)3.1
±0.2
0.2(3)8.8
±
±0.4 (8)4.5
±0.3 (6)4.7
±1.1 (6)
0 This ratio, the increase in pyrimidine incorporation obtained by inhibition of catabolism with DU, was not altered by
administration of purine nucleosides.
6 N.D., not determined.
Stimulation of '2 s IU incorporation by deoxyguanosine and
DU was observed in the spleen, liver, and small intestine, as
well as in cells of the IRC 741 leukemia (Table 2). In these
normal tissues, as in leukemic tissue, combined administration
of DU and deoxyguanosine increased the incorporation of
12SIU to that found with ' 2sIUdR.
Inhibition
of catabolism by DU administration
also
increases the incorporation of thymine into DNA (Table 3). At
doses of both 10 and 100 ¿tmoles/kg, the incorporation of
thymine is stimulated by DU and deoxyguanosine. At the high
dose, the incorporation of the base is elevated by DU and
deoxyguanosine to approximately 50% of that obtained with
thymidine. However, at a dose of 10 ¿/moles/kg,preferential
utilization of thymidine is observed. Administration of DU
and deoxyguanosine with a high dose of thymidine appears to
result in a small increase, approximately 40%, in thymidine
incorporation.
The increase in thymine incorporation resulting from
administration of DU is less than the increase obtained in IU
incorporation (Table 1). This may be related to differences in
metabolism of the 2 pyrimidines or to «utilization of labeled
catabolic products of 3H-labeled thymine which may not
readily
occur
with
12SIU (40).
Administration
of
methotrexate with 12SIU but not with 3H-labeled thymine
may also be a factor affecting the degree of stimulation
obtained with DU.
The incorporation of FU into RNA (Table 4) is also
stimulated by inhibition of catabolism with DU in the
leukemic and normal tissues studied. Both guanosine and
deoxyguanosine
produce only a minor stimulation of
incorporation. Since there is no specific stimulation by the
ribonucleoside, it appears that pyrimidine utilization in these
tissues is not limited by the availability of ribose 1-phosphate
or phosphoribosyl pyrophosphate for ribonucleotide synthesis.
The utilization of3 H-labeled uracil for nucleic acid synthesis is
similarly affected by DU and purine nucleosides in leukemic
tissue, small intestine, and liver.
394
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140i.s2
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120^
—¿r*~J-i
«o.E5loe—-PT:
l
15 30 45 60
i'„—
0 2.5 5.0"10
15
Fllint ut/Hi
1/11l/S 1/5 1/115/5
0/10 1/5 2/105/109/10
Tuie Deaths
Withoutn Miiiistutiii
«itkDu «dmiimtiilioii
Chart 4. Effect of DU on therapy of leukemia IRC 741 with FU.
Injections of DU (5 mg/kg) and varying doses of FU were administered
daily for 4 days beginning 1 day after unilateral s.c. tumor
implantation. Control animals not receiving FU received injections of
0.85% NaCl solution. Data are represented as the mean survival time of
animals that died of leukemia; percentage of controls ±S.D. The mean
survival time of control animals was 16 days and was not affected by
DU administration. The fraction of animals which died of chemical
toxicity represents animals which died between 7 and 14 days after
tumor implantation and showed no signs of leukemia on autopsy. In
the case of rats receiving DU and 15 mg/kg FU, the mean survival time
refers to the survival of the single rat not dying from chemical toxicity.
Systemic and Tissue-specific Catabolism. Table 5 illustrates
the lack of relationship between the specific activity of
dihydrouracil dehydrogenase in the 4 tissues investigated and
the stimulation of pyrimidine incorporation obtained with DU
in these tissues. In spite of the variation in catabolic activities,
CANCER RESEARCH VOL. 32
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In Vivo Inhibition offyrimidine
and particularly the absence of catabolism in leukemic tissue
and spleen, pyrimidine incorporation in all tissues is similarly
increased by DU administration. That there is no relationship
between tissue catabolic activity and increase in pyrimidine
utilization resulting from inhibition of catabolism suggests that
systemic catabolism, rather than catabolism in the target
tissue, is the primary factor that limits pyrimidine utilization
in vivo.
Tumor Therapy with FU. Since DU stimulates the
incorporation of FU into RNA, it is expected that inhibition
of catabolism will also potentiate the cytotoxicity of FU. The
experiments illustrated in Chart 4 demonstrate that both the
antitumor effects and the toxicity of FU are potentiated by
DU. The FU dose required to produce both toxicity and
tumor inhibition is decreased approximately 5-fold by DU
administration. DU itself has no toxic or antitumor effect at
the doses administered. At a given level of toxicity, similar
degrees of tumor inhibition by FU are observed whether or
not DU is administered, a fact indicating that inhibition of
catabolism with DU does not affect the chemotherapeutic
selectivity of FU.
DISCUSSION
We have previously described irreversible inhibition of
dihydrouracil dehydrogenase by DU in vivo and in a crude
enzyme preparation from rat liver (10). Inhibition of rat liver
dihydrouracil
dehydrogenase
by 5-cyanouracil has been
reported by Dorsett et al. (12), and this compound is also
active in inhibiting the catabolism of low doses (1.5 to 2.0
mg/kg) of uracil and FU in vivo (16). In contrast to DU,
5-cyanouracil is a reversible inhibitor (9, 10), and may
therefore be less adaptable to in vivo applications than DU,
particularly with regard to inhibition of the catabolism of large
doses of pyrimidines similar to doses administered in
chemotherapy and in this study. Administration of DU, 5.0
mg/kg, daily for a 4-day period is tolerated without significant
weight loss or gross signs of toxicity. The toxic effects of
larger doses of DU (chronic 50% lethal dose in Fischer rats is
10 mg/kg/day for 4 days) may be related to cytotoxic effects
of DU other than inhibition of pyrimidine catabolism (39,41,
42).
DU administration
results in a marked increase in the
utilization of pyrimidine bases for nucleic acid synthesis. This
effect of inhibiting catabolism is analogous to increasing
xanthine and hypoxanthine utilization by administration of
allopurinol, an inhibitor of xanthine oxidase (32, 33). On the
basis of our studies (8, 10), Ferdinandus and Weber (J.A.
Ferdinandus and G. Weber, personal communication) have
utilized DU and confirmed that inhibition of catabolism
increases thymine incorporation into DNA of developing rat
liver.
Combined administration
of DU and deoxyguanosine
increases the incorporation of high doses (100/mioles/kg of
12 5IU and 3H-labeled thymine into DNA to nearly the same
level of incorporation attained by administration of ' 2SIUdR
or 3H-labeled thymidine. This suggests that under these
conditions the low incorporation of the free base relative to
the deoxyribonucleoside results from catabolism of the free
Catabolism
base and the limited availability of deoxyribose 1-phosphate.
However, when the incorporation of thymine and thymidine is
compared at low doses (10 jumoles/kg) the incorporation of
thymidine is 10-fold higher than that of thymine even when
DU and deoxyguanosine are administered simultaneously. This
additional preference for deoxyribonucleoside
utilization at
low doses may be related to differences between nucleosides
and free bases regarding their mode of entry into cells.
Pyrimidine bases permeate cells by free diffusion (22), whereas
nucleosides are transported by a carrier-mediated mechanism
(23, 25, 29-31) and, once inside cells, may be trapped by
rapid phosphorylation (31). This would lead to a preferential
uptake of the nucleoside at low concentrations, at which
carrier-mediated transport is most active (23, 30), and
accounts
for preferential
incorporation
of thymidine
compared to thymine into DNA in vivo when low doses are
administered.
It might be anticipated that the incorporation of thymidine
or lUdR would be increased by administration of DU and
deoxyguanosine,
since these compounds would facilitate
reutilization of thymine or IU formed from phosphorolysis of
the administered deoxyribonucleosides. However, due to the
low efficiency of utilization of the free base compared to the
deoxyribonucleoside,
administration of DU and deoxygua
nosine would not be expected to increase deoxyribonucleoside
incorporation by more than a maximum of 50 to 75%. Such
an effect, which is of only marginal significance, is observed in
the case of thymidine incorporation. Studies on inhibition of
nucleoside phosphorolysis, in progress in our laboratory, may
elucidate the role of catabolism in pyrimidine nucleoside
utilization.
Although cells of the IRC 741 leukemia and the spleen have
no detectable catabolic activity, DU administration increases
pyrimidine incorporation in these tissues to the same extent as
in liver. This result implies that pyrimidine utilization in the
tumor and spleen is affected by circulating levels of precursors
which are limited by systemic catabolism occurring largely in
the liver. If only catabolism of pyrimidines in the tissue under
study limited their incorporation into nucleic acids, then DU
should affect pyrimidine utilization to a greater extent in
tissues with active catabolic pathways, such as the liver, than
in tissues lacking catabolic activity.
Pyrimidine catabolism is reduced or lacking in a number of
tumors studied (6, 7, 15, 21, 34-36) and shows a marked
negative correlation with growth rate in the Morris hepatoma
series (15, 35). Chaudhuri et al. (7) and Heidelberger (20) have
suggested that the lack of catabolic activity in tumors is
responsible for the chemotherapeutic
selectivity of FU.
However, the present results are not compatible with this
hypothesis, since the utilization of pyrimidines in tissues
lacking catabolic activity is controlled by systemic catabolism
in the liver. In order for a correlation to exist between the
tumor selectivity of FU and the lack of catabolism in tumors,
the role of catabolism in limiting FU utilization for RNA
synthesis and anabolism to fluorodeoxyuridylate would have
to be tissue specific rather than systemic. Futhermore, if
catabolism of FU in normal tissues but not in tumors was
significant to the chemotherapeutic
activity of FU, then
simultaneous administration
of DU should increase the
FEBRUARY 1972
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395
Geoffrey M. Cooper, W. F. Dunning, and Sheldon Gréer
systemic toxicity but not the tumor-inhibitory activity of FU.
However, we observe that combined administration of DU and
FU increases systemic toxicity and tumor inhibition to similar
extents.
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397
Role of Catabolism in Pyrimidine Utilization for Nucleic Acid
Synthesis in Vivo
Geoffrey M. Cooper, W. F. Dunning and Sheldon Greer
Cancer Res 1972;32:390-397.
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