Download Enhancement by Uridine of the Anabolism of 5

[CANCER RESEARCH
45, 4249-4256,
September 1985]
Enhancement by Uridine of the Anabolism of 5-Fluorouracil in Mouse
T-Lymphoma (S-49) Cells1
William B. Parker2 and Philip Klubes3
Department of Pharmacology,
The George Washington University School of Medicine, Washington, DC 20037
with FUra4 are related to the anabolism of the drug to FdUMP
ABSTRACT
Uridine enhances the growth inhibition due to 5-fluorouracil
(FUra) in a cultured mouse T-cell lymphoma (S-49). Using colony
formation assays we found that cytotoxicity produced by 24-h
continuous exposure to FUra (0.5 to 3.5 /¿M)
was increased more
than two-fold by simultaneous exposure to 10 /IM undine. Studies
were undertaken to explain the mechanism by which undine
enhanced the cytotoxicity of FUra in S-49 cells. Uridine (10 UM)
increased by about 50% both the anabolism of 1.0 pM [3H]FUra
to acid-soluble metabolites and the incorporation of 1.0 ^M [3H]FUra into RNA. However, the incorporation of 1.0 fiM [3H]FUra
into these fractions was less than that seen with 2.4 UM [3H]FUra, a dose which was equitoxic to 1.0 tiM [3H]FUra plus 10 ^M
and FUTP (1). Recently the incorporation of FUra into DNA (28) as well as the formation of fluorinated analogues of uridine
diphosphohexoses (9-13) have also been implicated in the cy
totoxicity of FUra. Ullman and Kirsch (14), using a mouse T-cell
lymphoma (S-49) cell line in culture, showed that simultaneous
administration of uridine with FUra decreases the concentration
of FUra required to inhibit cell growth. In S-49 cells FUra is
anabolized by OPRTase, and not by either uridine phosphorylase
or thymidine phosphorylase (15). Ullman and Kirsch (14) pro
posed that the enhancement by uridine of the action of FUra was
due to the formation of UTP from exogenous uridine, which
acted as a feedback inhibitor of de novo pyrimidine biosynthesis.
Presumably, the inhibition of de novo pyrimidine biosynthesis
decreases orotic acid levels and thereby increases the availability
undine. High-pressure liquid chromatography analysis of the
of PRPP and OPRTase for the anabolism of FUra to FUMP.
acid-soluble metabolites of FUra did not show any selective
Uridine has been shown to be effective as a modulator of the
change in specific FUra nucleotides, which could explain the
cytotoxicity of FUra. For example, uridine has been used as a
increased cytotoxicity associated with 10 ^M uridine. In addition,
rescue agent to increase the therapeutic index of FUra against
5-fluoro-2'-deoxyuridine
monophosphate levels and the amount
the mouse colon tumor 26 (16) and the mouse B16 melanoma
of [3H]FUra which was incorporated into the alkali-stable, acid(17). Protection from FUra toxicity afforded by the addition of
insoluble fraction were not increased by uridine. Uridine (10 ¿<M) uridine is apparently due to the reduction in the incorporation of
inhibited de novo pyrimidine biosynthesis by 70%, while 5- FUra into RNA rather than by reversal of FdUMP inhibition of
phosphoribosyl-1-pyrophosphate
levels were unchanged. Pre
thymidylate synthetase (18). Preliminary clinical trials using uri
dine infusion after FUra treatment are in progress (19-21).
sumably, the inhibition of de novo pyrimidine biosynthesis de
This study explored possible mechanisms by which uridine
creased orotic acid levels and allowed more FUra to be anabolmight enhance the action of FUra against S-49 cells in culture.
ized to 5-fluorouridine monophosphate via orotate phosphoriWe present evidence which supports the observation of Ullman
bosyl transferase. Furthermore, 2.4 /¿M
FUra inhibited the incor
and Kirsch (14) that uridine enhances the phosphoribosylation of
poration of [3H]deoxyguanosine into DNA by 50% after 24 h of
FUra. However, this increase in anabolism is restricted to the
incubation. In contrast, 1.0 I¿M
FUra plus 10 UM uridine did not
ribonucleotide pool and can account for, at most, only 50% of
inhibit the incorporation of [3H]deoxyguanosine into DNA. The
the increased cytotoxicity. A preliminary report of our investiga
data suggested that there was a qualitative difference in the
tion has already appeared (22).
mechanism by which 1.0 UM FUra plus 10 MMuridine killed S-49
cells as compared to 2.4 ¿¿M
FUra alone, and that the enhance
ment by uridine of the cytotoxicity of FUra was due, in part, to
the increased anabolism of FUra to ribonucleotides.
MATERIALS AND METHODS
Chemicals and Supplies. FUra, MIX. and PALA were obtained from
the Drug Synthesis and Chemistry Branch, National Cancer Institute. 5Fluoro-2'-deoxyuridine,
5-fluorocytidine, and 5-fluorouridine were ob
tained as gifts from Hoffmann-La Roche, Inc. (Nutley, NJ). 5-Fluoro-2'-
INTRODUCTION
Severe disruptions in DNA and RNA synthesis after treatment
1This investigation was supported by Grant CH-160 from the American Cancer
Society, and Training Grant T32-CA09223 from the National Cancer Institute,
Department of Health and Human Services.
2 From a dissertation presented to the Department of Pharmacology, The
Graduate School of Arts and Sciences, The George Washington University, in
partial fulfillment of the requirements for the Ph.D. degree. Present address:
Department of Pharmacology, 916 FLOB 231-H, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27514.
9 To whom requests for reprints should be addressed, at the Department of
Pharmacology, The George Washington University Medical Center, 2300 Eye
Street, NW, Washington, DC 20037.
Received 11/30/84; revised 5/2/85; accepted 5/13/85.
deoxycytidine was obtained from Calbiochem (La Jolla, CA). 5-Fluorocytosine was a gift from Dr. G. Wagner (The George Washington
University Medical School, Washington, DC). FUMP and FUTP were
obtained from Sierra Bioresearch (Tucson, AR). FdUMP was obtained
from Terra Marine Bioresearch (La Jolla, CA). Crude yeast OPRTase and
' The abbreviations used are: FUra, 5-fluorouracil; FdUMP, 5-fluoro-2'-deoxyuridine monophosphate; PRPP, 5-phosphoribosyl-1 -pyrophosphate; FUMP, 5-fluo
rouridine monophosphate; FUTP, 5-fluorouridine triphosphate; UTP, uridine triphosphate; PALA, W-<phosphonacetyl)-L-aspartate; MTX, methotrexate; OPRTase, orotate phosphoribosyl transferase; TCA, trichtoroacetic acid; HPLC, high-pressure
liquid chromatography; FUrd, fluorouridine; FdUrd, fluorodeoxyuridine; FUDP, 5fluorouridine diphosphate; FCyt, 5-fluorocytosine; FCyd, 5-fluorocytidine; FdCyd,
5-fluorodeoxycytidine.
CANCER RESEARCH VOL. 45 SEPTEMBER
1985
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LIBIDINE ENHANCEMENT
orotidine-5'-phosphate
decarboxylase, and Crotalus atrox snake venom
were obtained from Sigma Chemical Company (St. Louis, MO). [6-3H]FUra (18 Ci/mol) was obtained from Moravek Biochemicals (City of
Industry, CA). [5-3H]Orotic acid (20 Ci/mol), [14C]NaHCO3 (46 mCi/mmol),
and [2,8-3H]adenosine (35.2 Ci/mmol) were obtained from New England
Nuclear (Boston, MA). [8-3H]Deoxyguanosine (7.8 Ci/mmol) and [4,5-3H]-
OF FUra ANABOLISM
The remaining pellet, which contained the alkali-stable, acid-insoluble
fraction, was washed twice with 1.0 ml of ice-cold 5% TCA, and was
dissolved in 1.0 ml of 0.3 N NaOH. A 0.5-ml aliquot of each sample was
mixed with 10 ml of Liquiscent (National Diagnostics, Somerville, NJ),
and the radioactivity was measured in a Beckman LS 6800 liquid
scintillation counter. Results were corrected for counting efficiency with
an external standard and results were expressed as dpm/107 cells.
leucine (58 mCi/mmol), were obtained from ICN Pharmaceuticals (Irvine,
CA). All other chemicals were standard analytical grade.
Growth Conditions for S-49 Cells. S-49 cells, obtained from Dr. J.
Authentic standards of the FUDP-hexoses were not available. In each
experiment two radioactive peaks eluted from the column just after
FdUMP. The quantity and location of these peaks were similar to those
reported for FUDP-hexoses by Pogolotti ef al. (24). Therefore these
peaks were assumed to be FUDP-hexoses (presumably FUDP-glucose
and FUDP-galactose).
In order to identify the [3H]FUra anabolites with retention times greater
Maybaum at the University of California School of Medicine, San Fran
cisco, were grown as suspension cultures in Dulbecco's modified Eagle's
medium (GIBCO, Grand Island, NY) supplemented with 2.2 g NaHCO3/
liter, 2 g glucose/liter, and 10% heat inactivated horse serum (GIBCO),
pH 7.4. The cells were kept at 37°Cin a 5% CO2-95% O2 atmosphere.
Fresh cells were thawed every 9 months during the course of the study
to maintain genetic stability in the culture. The cells were assayed by
Biofluids, Inc. (Rockville, MD) and were found to be free from Myco-
than 55 min (i.e., the di- and triphosphates),
the radioactive peaks were
plasma contamination.
Effect of Drug Treatment on Cell Growth and Viability. Exponentially
growing S-49 cells were added to media containing the desired concen
tration of drug(s). Most experiments were begun with 1 x 105 cells/ml.
All experiments were carried out in 25-cm2 Falcon culture flasks. One ml
atrox snake venom/ml in 0.06 M Tris:0.02 M MgCI2, pH 9. The protein
was precipitated by the addition of 50 n\ ice-cold TCA (100 g dissolved
collected as they eluted off the column and lyophilized. The samples
were then incubated overnight at 37°C with 1.0 ml of 20 mg Crotalus
of cell suspension was removed from each flask at the time indicated
(usually at 24, 48, and 72 h after drug addition), and cell numbers were
counted with a Model Zf Coulter Counter. The data were presented as
percentage of control cell growth.
To measure viable cells after drug treatment, S-49 cells were cloned
in a 0.5% agar solution which contained 20% horse serum and 40%
conditioned medium (23). Conditioned medium was obtained from ex
ponentially growing S-49 cell suspensions (0.5-1.0 x 106 cells/ml) by
centrifugation at 250 x g for 5 min, followed by filtration through a 0.22
urn filter. After 12 to 14 days, colonies (greater than 50 cells) were visible
to the naked eye and could be counted. The cloning efficiency of S-49
cells was about 50%.
Measurement of the Acid-soluble Metabolites of FUra in S-49 Cells.
The method of Pogolotti ef a/. (24) was used to separate the acid-soluble
metabolites of FUra. After the addition of drugs, each cell suspension
contained approximately 1 x 106 cells/ml in 100 ml of culture medium.
The S-49 cells were incubated with 1.0 »M[3H]FUra (1.125 Ci/mmol),
1.0 IM [3H]FUra (1.125 Ci/mmol) plus 10 »Muridine, or 2.4 MM[3H]FUra
(1.125 Ci/mmol). After 6 h of incubation, two 45-ml aliquots from each
treatment group were centrifugea at 250 x g for 5 min at room temper
ature to harvest the cells. The cell pellets were washed once with icecold phosphate-buffered saline, and the acid-soluble metabolites were
extracted with 0.25 ml of ice-cold 0.6 M TCA containing 0.10 mg/ml each
of FUra, FUrd, FdUrd, FUMP, FdUMP, and FUTP. The samples were then
mixed with 0.25 ml of 0.5 M tricaprylyl tertiary amine in trichlorotrifluoroe-
in water to 100 ml). TCA was extracted by mixing with 1.0 ml of 0.5 M
tricaprylyl tertiary amine in trichlorotrifluoroethane
and centrifuging at
1000 x g for 5 min. The aqueous layer was collected, evaporated under
vacuum at 43°C, and resuspended in 250 n\ of water plus 50 ¡Aof
solution containing FUra, FCyt, FUrd, FCyd, FdUrd, and FdCyd (0.16
mg/ml each). Twenty ^l of the sample were counted for radioactivity and
200 n\ were analyzed for fluoropyrimidine bases and nucleosides using
a Waters C-18, radial compression lO-^m HPLC column. The mobile
phase was 10 ITIMsodium acetate, pH 4.5, with a flow rate of 2.0 ml/
min.
Measurement of Intracellular PRPP Levels. The method of May and
Krooth (26) was used, with some modification, to measure intracellular
PRPP. Approximately 2 x 10s exponentially growing S-49 cells were
treated with 10 MMuridine, 300 /¿M
PALA, 0.04 IM MTX, or 0.4 MMMTX
for 6 h. The cell pellet, which was collected by centrifugation at 250 x g
for 5 min at 4°C,was mixed in 1.0 ml of water and disrupted by sonication
at 4°C.The supernatant fluid (0.9 ml) was collected after centrifugation
(18,000 x g for 5 min at 5°C)and was assayed for PRPP. The assay
utilized OPRTase and orotidine-5'-phosphate
decarboxylase to convert
[3H]orotic acid to [3H]uridylate. PRPP is made rate limiting in this reaction,
and the amount of [3H]uridylate that is formed is equal to the amount of
PRPP that is present. [3H]Uridylate was separated from [3H]orotic acid
by paper chromatography (26). The [3H]uridylate spot was cut from the
paper and placed in a scintillation vial with 10 ml of Liquiscent, and the
radioactivity was determined by extrapolation from a standard curve
(nmol PRPP added versus [3H]UMP formed) which was prepared for
thane to extract the TCA (25). Two hundred n\ of this solution were
injected onto a Waters C-18 radial compression 10-MM HPLC column
each experiment.
Measurement of the Rate of de Novo Pyrimidine Biosynthesis. The
method of Karle ef al. (27) was used to determine the effect of drug
treatment on de novo pyrimidine biosynthesis. S-49 cells (5x106) were
(Milford, MA). During each analysis two solvents were mixed by a Waters
Associates Model 660 solvent programmer according to a concave
upward program (Curve 8). Solvent A contained 5 mw tetrabutylammon-
incubated with 10 UM uridine, 1.0 /¿MFUra, 1.0 JIM FUra plus 10 UM
uridine, or 2.4 ^M FUra for 6 h in 5 ml of Dulbecco's modified Eagle's
medium which contained 6.5 mM NaHCO3. Twenty-five ^Ci of [14C]-
ium hydrogen sulfate and 5 ITIM potassium phosphate, pH 7. Solvent B
is a mixture of Solvent A plus methanol (60/40, v/v). Solvent A was
replaced entirely by Solvent B over a 60-min period by the solvent
programmer, followed by a 10-min run with Solvent B. The column was
NaHCOs were added to each incubation during the last hour. At the end
of the incubation period, the cells were collected by centrifugation at 150
x g for 5 min, and the macromolecules were precipitated with ice-cold
reequilibrated with Solvent A for 10 min before another injection. The
flow rate was 2 ml/min. FUra and its metabolites were detected by their
absorbance at 254 nm.
To measure the incorporation of [3H]FUra into RNA and the alkali-
5% TCA. The TCA was extracted by mixing with 0.5 M tricaprylyl tertiary
amine in trichlorotrifluoroethane,
and the uridine nucleotides were de
graded to uridine by Crotalus atrox snake venom. Pyrimidine nucleosides
were added to each sample for detection purposes, and they were
separated using a Waters C-18 radial compression 10 ^m HPLC column
stable, acid-insoluble fraction, the TCA precipitate of the cell extracts
was washed twice with 1.0 ml of ice-cold 5% TCA, and resuspended in
3 ml of 0.3 N NaOH. After incubation in a 37°Cshaking water bath for 3
(Milford, MA). The mobile phase was 10 mM sodium acetate, pH 4.5,
flow rate 2.0 ml/min. The nucleosides were detected by their absorbance
at 254 nm. Fractions containing each pyrimidine (uridine, cytidine, de-
h, 300 ¿ilof ice-cold 5.2 N HCIO«were added, and the samples were
centrifugea at 18,000 x g for 5 min at 5°C.The supernatant fluid from
oxycytidine, or deoxyuridine) were collected from the column, and the
radioactivity was determined as described above. In control cells the
incorporation of [14C]NaHCO3 into uridine nucleotides was linear with
each sample was removed and counted for radioactivity (RNA fraction).
CANCER RESEARCH
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1985
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URIDINE ENHANCEMENT
OF FUra ANABOLISM
respect to time over a 90-min period.
Measurement of Intracellular DTP Levels. Thirty-five ml of S-49 cells
(7.14 x 105 cells/ml) were incubated with 1.0 //M FUra, 2.4 ¿IM
FUra, 10
MMundine, 1.0 UM FUra plus 10 ^M uridine, or no drug. After 6 h the cells
from each treatment group were collected by centrifugaron and washed
with 1.0 ml of ice-cold phosphate-buffered saline, pH 7.4. Cells were
Õ
80-
harvested by centrifugation, and the cell pellets were then mixed with
0.5 ml of ice-cold 10% TCA. The acid-insoluble material was removed
by centrifugation and the acid-soluble extract was mixed with 0.5 ml of
0.5 M tricaprylamine in trichlorotrifluoroethane
to remove TCA. The
suspensions were centrifuged at 1000 x g for 5 min at 4°Cand 0.4 ml
Fln pl.s
10«*Uni
of the aqueous layer was removed and filtered through a 0.45-^m
Millipore filter. Samples were stored at -70°C until they could be
analyzed by HPLC. The nucleoside triphosphates were separated using
a 10-Ã-Ã-Ã-Ti
SAX column in a radial compression HPLC system (Waters
Associates, Milford, MA). The mobile phase was 0.4 M Na2HPO4, pH 3.6,
at a flow rate of 2.0 ml/min. The amount of UTP in each sample was
determined by extrapolation from a standard curve relating UTP peak
height to the concentration of UTP.
Measurement of Nucleic Acid Synthesis. Cells were incubated with
no drug, FUra (1.0 or 2.4 ^M), 1.0 ¿JM
FUra plus 10 MMuridine, or 10 MM
uridine alone for 2, 6, or 24 h. One h before the end of each incubation,
1 fiCi/ml of either [3H]adenosine or [3H]deoxyguanosine was added to
each cell suspension. Thirty min before the end of each incubation two
0.25-ml aliquots were removed and the cell numbers were determined
using a Model Zf Coulter Counter. At the end of each incubation two 2-
00
25
05
10
20
FUra (,»)
Chart 1. Enhancement by uridine (Urd) of the growth inhibition due to FUra. S49 cells (1 x 10* cells/ml) were incubated with FUra (0.125-2.0 ¡iu)either atone or
with 10 fiu undine. After 72 h of incubation, two 10-m l aliquots were obtained
from each treatment and cell numbers were determined using a Model Zf Coulter
Counter. The original cell number was subtracted from the 72-h cell count of each
treatment and the results were presented as a percentage of control growth. There
were approximately 1.3 x 10°cells/ml in control flasks after the 72-h incubation.
Points, mean of three experiments; oars, SE.
ml aliquots were transferred from each cell suspension to microfuge
tubes containing 2.0 ml of ice-cold 10% TCA. These samples were
frozen until further analysis.
Each sample was thawed and centrifuged at 1000 x g for 5 min at
room temperature. The supernatant fluid from each sample was dis
carded and the pellets were washed twice with ice-cold 0.2 N HCIO4.
The pellets were incubated for 2 h in 0.3 N NaOH at 37°C, and then 0.3
ml of 5.2 N HCIO4 was added. The suspensions were centrifuged, and
the pellets were washed twice with 1.0 ml of 0.2 N HCIO4. The super
natant fluid and two washes were combined and an aliquot was mixed
with 10 ml of Liquiscent. The radioactivity was then determined as
described above. This fraction represented the incorporation of [3H]adenosine or [3H]deoxyguanosine into RNA. The pellet was dissolved in
1.0 ml of 0.3 N NaOH and transferred to a scintillation vial. The solutions
were acidified with 0.1 ml of 100% TCA and counted for radioactivity in
10 ml of Liquiscent. This fraction represented the incorporation of [3H]adenosine or [3H]deoxyguanosine into DNA.
In control cells the incorporation of either [3H]deoxyguanosine or [3H]
adenosine into either RNA or DNA was linear with respect to time over
a 90-min period.
RESULTS
100-1
FUra
o
110-
2
o
FUra pi» lOpM Urd
z
o
o
2
HI
O
C
01-
Effect of Undine on the Cytotoxicity of FUra. Ullman and
Kirsch (14) reported that the concentration of FUra required to
inhibit the growth of S-49 cells by 50% was decreased from 0.8
to 0.5 UM in the presence of 50 ^M uridine. We examined the
effect of various concentrations of uridine (2.5 to 100 ^M) to
increase the action of FUra against S-49 cells. Uridine (10 ^M)
produced the maximum enhancement of cell growth inhibition
due to FUra (data not shown). In addition, 10 /¿M
uridine alone
did not affect cell growth. Therefore, in all further experiments,
10 ¿¿M
uridine was used with FUra.
The effect of 10 pu uridine on the inhibition of cell growth by
various concentrations of FUra was examined. As can be seen
(Chart 1), 10 MM uridine decreased the concentration of FUra
required to inhibit the growth of S-49 cells by 50% from 0.73 to
0.4 MM(P < 0.01, Student's t test).
CANCER
RESEARCH
0.5
1.0
1.5
2.0
2.5
I
30
FUraOiM)
Chart 2. Effect of uridine (Urd) on the cloning efficiency of cells treated with
FUra. S-49 cells (1 x 105 cells/ml) were incubated with FUra (0.5-3.0 t¡u)either
alone or with 10 /¿M
uridine. After 24 h, cells from each treatment were collected
and cloned. Twelve to 14 days later the colonies were counted. The cloning
efficiency from each treatment was determined and the results are presented as
percentage of control cloning efficiency The cloning efficiency of control cultures
was about 50%. Points, mean of three experiments; oars, SE.
The effect of drug treatment on colony formation is a more
sensitive indicator of cell kill than is the inhibition of cell growth.
Therefore, S-49 cells were treated with various concentrations
of FUra (0.5 to 3.0 MM),with or without 10 MM uridine for 24 h
and cloned in nutrient agar (Chart 2). Uridine alone had no effect
on cell viability. However, uridine decreased the concentration of
FUra required to kill 50% of the cells from 1.85 MMto 0.7 MM.
VOL. 45 SEPTEMBER
1985
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URIDINE ENHANCEMENT
OF FUra ANABOLISM
MM [3H]FUra plus 10 MM uridine, or 2.4 MM [3H]FUra, and the
intracellular levels of FUra, its anabolites, and its incorporation
into RNA were determined (Chart 3). There was no difference in
the amount of FUra in cells treated with either 1.0 MM [3H]FUra
or 1.0 MM [3H]FUra plus 10 MM uridine. In contrast, 2.4 MM [3H]-
OfjM »H-FUra
Qi
,¡v»HFUra
30-20-10-r1TREATMENTDi
URO•
plus lOuM
'H-FUra|M
.:,.'-'
FUra increased the level of intracellular FUra approximately 150%
as compared to 1.0 MM[3H]FUra. Similarly, 2.4 MMFUra increased
the levels of FUrd, FUMP, FdUMP, FUDP-hexoses, and FUTP as
compared to 1.0 MM [3H]FUra. Treatment with 1.0 MM [3H]FUra
plus 10 MMuridine increased the levels of the FUDP-hexoses and
FUra
FUrd
À
JÕ
FUMP
FdUMP
FUTP as compared to 1.0 MM FUra alone. There were trends,
which were not statistically significant, toward increases in FUrd,
FUMP, FdUMP, and FUDP after treatment with 1.0 MM[3H]FUra
plus 10 MM uridine as compared to 1.0 MM [3H]FUra alone.
\\300-200-100-+rffÃ-fI*
FUDP
FUDP
MEXOSES
FUTP
RNA
Chart3. Effect of undine (URD) on the anabolism of FUra to acid-soluble
metabolites. S-49 cells (1 x 10e cells/ml) were incubated with 1.0 /IM [3H]FUra, 1.0
/IM [3H]FUra plus 10 AIMuridine, or 2.4 /IM (3H]FUra. After 6 h, two 45-ml aliquots
were collected from each treatment and the acid-soluble metabolites of FUra as
well as its incorporation into RNA was determined. Columns, mean of three
experiments; oars, SE. *, significantly different from either 1.0 /JM |3H]FUra alone,
or 1.0 itM [3H]FUra plus 10 »IM
uridine, P < 0.05; Newman Keuls' multiple range
test. +, significantly different from 1.0 /IM [3H]FUra, P < 0.05; Newman Keuls'
Furthermore, 10 MM uridine did not increase the anabolism of
any acid-soluble metabolite of 1.0 MM[3H]FUra to the level seen
with 2.4 MM [3H]FUra. After 24 h of incubation, the effect of 10
MMuridine on the anabolism of 1.0 MM [3H]FUra to its individual
metabolites
was qualitatively
similar to that seen in the 6-h
incubation (data not shown).
Uridine (10 MM) also increased the incorporation of 1.0 MM
[3H]FUra into RNA by 40% as compared to 1.0 MM [3H]FUra
multiple range test.
alone; however, this incorporation was less than that seen with
2.4 MM [3H]FUra alone (Chart 3). Uridine (10 MM)caused similar
increases in the incorporation of 1.0 MM[3H]FUra into RNA after
2, 4, or 24 h of incubation. In addition, at these time points the
amount of [3H]FUra in RNA of cells treated with 1.0 MM[3H]FUra
plus 10 MMuridine was also less than that seen with 2.4 MM[3H]FUra (data not shown).
The FUDP and FUTP peaks were collected to confirm the
identity of the radioactivity associated with these peaks. The
nucleotides were degraded to nucleosides by Crotalus atrox
snake venom, and rechromatographed in a HPLC system used
to separate fluoropyrimidine nucleosides. More than 85% of the
radioactivity coeluted with FUrd, indicating that the radioactivity
in the FUDP and FUTP peaks was due to [3H]FUDP and [3H]Chart 4. Effect of uridine (Urd) on the incorporation of [3H]FUra into the alkalistable, acid-insoluble fraction. S-49 cells (1 x 10e cells/ml) were incubated wth 1.0
,,M [3H]FUra, 1.0 nu [3H]FUra plus 10 JIM uridine, or 1.7 nM [3H]FUra for 24 h. At
0, 2, 4, 6, and 24 h two 45-ml aliquots from each treatment were collected and the
radioactivity in the alkali-stable, acid-insoluble fraction was determined. Points,
mean of three experiments; bars, SE. Absence of SE bars indicates that the SE
was less than the size of the symbol. *, significantly different from cells treated
with either 1.0 *iM [3H]FUra plus 10 ^M uridine or 1.0 »u[3H]FUra alone, P < 0.05;
Newman Keuls1 multiple range test.
These results, which were consistent with the growth inhibition
experiments (Chart 1), indicated that 10 UM uridine enhanced the
cell-killing action of FUra by about 2.5-fold. FUra (1.0 MM)alone
did not decrease cell viability, whereas either 1.0 MM Fura plus
10 MM uridine or 2.4 MM FUra alone were equitoxic in that they
both decreased cell viability approximately 85%.
Effect of Undine on the Anabolism of FUra. One mechanism
by which uridine could enhance the cytotoxicity of FUra would
be to increase the anabolism of FUra to active nucleotides (14).
To test this hypothesis we determined the effect of uridine on
the anabolism of FUra to acid-soluble metabolites, as well as its
incorporation into RNA and into an alkali-stable, acid-insoluble
fraction. The alkali-stable, acid-insoluble fraction should consist
of [3H]FdUMP bound to thymidylate synthetase and [3H]FUra
incorporation into DNA (28, 29).
S-49 cells were incubated for 6 h with 1.0 MM [3H]FUra, 1.0
CANCER
RESEARCH
FUTP, respectively. No radioactivity was detected in the fractions
corresponding to FCyd, FdCyd, or FdUrd, which indicated that
their di- or trinucleotides were not formed. This method was
sensitive enough to detect 0.13 pmol of a fluorinated nucleotide
(data not shown).
Uridine (10 MM) did not increase the incorporation of 1.0 MM
[3H]FUra into the alkali-stable, acid-insoluble fraction of cells
(Chart 4). The alkali-stable, acid-insoluble fraction should consist
of the thymidylate synthetase: FdUMP:/v5,/V10-methylenetetrahydrofolate complex plus the incorporation of [3H]FUra into DNA
(28, 29). Since the incorporation of [3H]FUra into DNA accounts
for only a small percentage (less than 3%) of the amount of
radioactivity found in this fraction,5 this fraction primarily repre
sents FdUMP which is bound to thymidylate synthetase. Fur
thermore, the results seen in Chart 4 are consistent with the
observation (Chart 3) that the formation of [3H]FdUMP was
similar in cells treated with either 1.0 MM [3H]FUra plus 10 MM
uridine or 1.0 MM[3H]FUra alone.
Effect of Uridine on the Level of PRPP. It has been hypoth
esized that uridine enhances FUra action due to its conversion
to UTP and a subsequent feedback inhibition by UTP of de novo
pyrimidine biosynthesis (14). Decreases in the intracellular levels
5W. B. Parker, unpublished observation.
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1985
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URIDINE ENHANCEMENT
OF FUra ANABOLISM
of orotic acid should increase PRPP which is necessary for the
120conversion of FUra to FUMP by OPRTase (30). The increased
anabolism of FUra could then contribute to, or account for, the
increased cell kill seen with this combination.
100-O
To test this hypothesis, the effect of uridine on intracellular
PRPP levels was measured. S-49 cells were incubated for 6 h
1C£
with either 10 MM uridine, 300 MM PALA, 0.04 MM MTX, 0.4 MM 80-O
MTX, or no drug, and intracellular PRPP levels were measured.
üLU°
In five experiments which were carried out, 10 MMuridine did not
60-Z
increase intracellular PRPP levels (data not shown). Since uridine
did not affect PRPP levels, the effect of either PALA or MTX on LUO
PRPP levels was examined to confirm that increases in PRPP or
UJ
40-1
could be detected. PALA (300 MM)or 0.40 UM MTX both caused
o.20a 90% decrease in growth of S-49 cells over a 72-h period (data
not shown), which was similar to that of 1.0 MMFUra plus 10 MM
uridine (Chart 1). However, neither PALA (300 MM)nor MTX (0.04
MM)increased intracellular PRPP levels in S-49 cells. However,
0.4 MMMTX did increase intracellular PRPP levels.
0T*URDTT*FUra
FUra(10,iM)
FUra +URD
One experiment was carried out to determine the effect of
(1.0MM)
(1.0„M;10»iM) (24/jM]
either 10 MMuridine, 1.0 MMFUra, 1.0 MMFUra plus 10 MMuridine,
Charts. Effect of uridine (URD) on the incorporation of ["C]NaHC03
into
or 100 MM uridine on intracellular PRPP levels. Only 100 MM individual pyrimidine nucleotides. S-49 cells (1 x 10* cells/ml in 5 ml) were incubated
in the presence of FUra (1.0 or 2.4 ^M). 10 ^M uridine, or 1.0 ^M FUra plus 10 ¡it*
uridine appeared to increase intracellular PRPP levels (148% of
uridine. After 5 h of incubation, 25 ß\of ['4C)NaHC03 were added to each treatment
control), whereas 10 MM uridine, 1.0 MM FUra, or 1.0 MM FUra group. After an additional 1 h of incubation the incorporation of ["CINaHCOs into
acid-soluble pyrimidine nucleotides was determined. The data are presented as
plus 10 MM uridine had little, if any, effect on intracellular PRPP
percentage of control pyrimidine synthesis. Columns, mean from three experiments,
levels (111, 113, and 122% of control, respectively; data not
except for 2.4 ,.M FUra. which is the result from only one experiment; oars, SE. *,
significantly different from control, P < 0 05; Newman Keuls' multiple range test.
shown).
Effect of Undine on de Novo Pyrimidine Biosynthesis. The
observation that 10 MM uridine failed to increase intracellular
Table 1
Comparison of FUTP and UTP levels, and the
RNAnmol/107
incorporation of FUra into
PRPP levels did not support the concept that uridine enhanced
cellsTreatment1
the cytotoxicity of FUra by increasing PRPP levels and thereby
increasing the anabolism of FUra. Therefore, the effect of uridine,
x
FUra, or FUra plus uridine on de novo pyrimidine biosynthesis
1000.94(1.0)1.14(1.2)2.25
was examined. As can be seen (Chart 5) treatment of cells with
.0FUra10
/IM
(1 .Of
.0)0.045(1.8)0.061
(1
either 10 MMuridine or 1.0 MMFUra plus 10 MMuridine decreased
3.91
(1.5)2.71
0.085(1.4)0.138(2.3)FUTP:UTPratio
the incorporation of [14C]NaHC03 into total pyrimidine nucleolM uridine
2.4 MMFUraUTP"2.67
(1.0)FUTP00.025(2.4)FUra-RNA00.060(1.0)
(2.4)
" Values were obtained from Chart 7.
6 Values were obtained from Chart 3.
c Numbers in parentheses, ratio of drug treatment to treatment with 1.0 »uFUra
tides to 30% of control. In contrast, FUra (either 1.0 or 2.4 MM)
alone had no effect on the incorporation of [14C]NaHCO3 into
pyrimidine nucleotides.
Effect of Undine on UTP Levels. Presumably the inhibitionof
de novo pyrimidine biosynthesis by uridine (Chart 5) was due to
increase in intracellular UTP, which acts as a feedback inhibitor
of carbamoyl phosphate synthetase II (30). In order to determine
if intracellular UTP levels were increased, UTP was measured
after incubation of S-49 cells for 6 h with 10 MMuridine, 1.0 MM
FUra, 2.4 MM FUra, or 1.0 MM FUra plus 10 MM uridine. None of
these treatments changed intracellular UTP levels (data not
shown). However, while controls had 2.54 nmol of UTP/107 cells,
treatment with either 1.0 MM FUra plus 10 MM uridine or 10 MM
uridine alone resulted in 4.00 nmol of UTP/107 cells. Although
the increases in UTP due to either of these treatments were not
shown to be statistically significant, the data suggested a trend
toward increased UTP levels which might account for the ob
served inhibition in de novo pyrimidine biosynthesis (Chart 5).
Comparison of FUTP, UTP, and Incorporation of FUra into
RNA. A comparison of the effect of uridine on UTP levels, FUTP
levels, and the incorporation of FUra into RNA can be seen in
Tabble 1. FUra (2.4 MM)increased both the levels of FUTP and
its incorporation into RNA about 2.4 times as compared to cells
treated with 1.0 MM FUra (Chart 3; Table 11 This indicated that
when cells were treated with FUra alone the fer .-nation of FUra-
CANCER
alone, which is taken as 1.0.
RNA correlated with the levels of FUTP. FUra (2.4 MM) had no
effect on intracellular UTP pools (Table 1). In cells treated with
1.0 MMFUra plus 10 MMuridine, the FUTP levels were 1.8 times
that seen in cells treated with 1.0 MMFUra alone (Chart 3; Table
1). However, the incorporation of FUra into RNA in cells treated
with the combination was only 1.4 times that of cells treated with
1.0 MM FUra alone. Treatment with 1.0 MM FUra plus 10 MM
uridine resulted in less incorporation of FUra into RNA than
would be expected based on FUTP levels. It is possible that the
trend toward increased UTP after treatment with 1.0 MM FUra
plus 10 MMuridine accounted for the decreased incorporation of
FUTP into RNA. Thus, the formation of FUra-RNA was more
closely related to the FUTP: UTP ratio than it was with FUTP
levels.
Effect of Drug Treatment on Macromolecular Synthesis.
The results shown above (Chart 3) indicated that uridine in
creased the anabolism of 1.0 MM FUra to levels which were
approximately halfway between those seen with 1.0. MM FUra
and 2.4 MM FUra (the latter of which was equitoxic to 1.0 MM
FUra plus 10 MMuridine; see Chart 2). Therefore, the increased
RESEARCH VOL. 45 SEPTEMBER
1985
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URIDINE ENHANCEMENT
anabolism of FUra may not account for all of the enhanced
cytotoxicity of the combination of 1.0 MMFUra plus 10 MMuridine.
In a further attempt to localize the site at which uridine enhances
the cytotoxicity of FUra, the effect of either FUra or FUra plus
uridine on protein, RNA, and DMA synthesis was determined.
Incubation of S-49 cells for 2, 6, or 24 h with 1.0 MMFUra, 2.4
MM FUra, or 1.0 MM FUra plus 10 MM uridine did not affect the
incorporation of either [3H]leucine into total protein or [3H]adenosine into total RNA (data not shown). However, FUra (2.4 MM)
inhibited the incorporation of [3H]deoxyguanosine into DNA by
50% after 24 h of incubation, respectively (Chart 6). In contrast,
during 24 h of incubation, neither 1.0 MM FUra nor 1.0 MM FUra
plus 10 MM uridine inhibited DNA synthesis. It was of interest
that at 24 h this result was qualitatively different from that
obtained with 2.4 MMFUra. This suggested that the mechanism
of cell kill for 1.0 MM FUra plus 10 MM uridine might be different
from that of the equitoxic dose of FUra (2.4 MM)alone.
DISCUSSION
Enhancement by Undine of the Anabolism of FUra. Ullman
and Kirsch (14) reported that uridine enhances both the growth
inhibitory effects of FUra against S-49 cells, as well as the
phosphoribosylation of FUra. They suggested, but did not at
tempt to prove that uridine increases the action of FUra by
increasing its anabolism. Our data (Chart 1) confirmed their
observation, and in addition, we showed that uridine enhanced
the cell killing action of FUra by about 2.5-fold, as determined by
colony formation assay (Chart 2). It is of interest that the con
centration of uridine in the serum of mice is approximately 10 MM
(31), and the concentration of uridine in human serum ranges
from 2 to 9 MM (32). The results reported herein indicate that
physiological levels of uridine can affect the anabolism and action
of FUra. Furthermore, these results suggest that in in vitro
studies it may be more meaningful to study the mechanism of
action of the antimetabolites in the presence of relevant physio
logical metabolites.
In order to test the hypothesis that uridine increases the
OF FUra ANABOLISM
cytotoxicity of FUra by increasing the anabolism of FUra to
FUMP, the effect of uridine on the anabolism of FUra to active
nucleotides was examined. Uridine (10 MM) increased both the
anabolism of 1.0 MM FUra to acid-soluble metabolites and the
incorporation of FUra into RNA (Chart 3). If the increase in
cytotoxicity due to uridine was solely due to increased anabolism
of FUra, then the levels of the acid-soluble metabolites of FUra
as well as the amount of FUra incorporated into RNA in cells
treated with 1.0 MM FUra plus 10 MM uridine should be equal to
that of cells treated with 2.4 MMFUra. However, these increases
in FUra anabolism due to 10 MM uridine were not equal to the
levels seen with 2.4 MM FUra alone (Chart 3). Therefore, the
increase in FUra anabolism due to uridine, which was observed,
did not appear to account for all of the increase in the cytotoxicity
of 1.0 MMFUra plus 10 MMuridine.
The effect of uridine on the formation of indvidual anabolites
of FUra was studied to determine if uridine selectively increased
any particular FUra nucleotide. After treatment with 1.0 MMFUra
plus 10 MM uridine, both FUTP and the FUDP-hexoses were
increased above the levels in cells treated with 1.0 MM FUra
alone, while the other acid-soluble metabolites showed a trend
(which was not statistically significant) toward increases (Chart
3). Furthermore, in cells treated with 1.0 MM FUra plus 10 MM
uridine the levels of each of the acid-soluble metabolites of FUra
(including FUTP, FdUMP, and FUDP-hexoses) were significantly
less than from cells treated with 2.4 MM FUra alone. Therefore,
uridine did not selectively increase the formation of any particular
acid-soluble metabolite of FUra to a level which could account
for the increase in cytotoxicity of this combination.
Uridine did not affect the incorporation of FUra into the alkalistable, acid-insoluble fraction of S-49 cells (Chart 4). Most (ap
proximately 97%) of the radioactivity in this fraction consists of
the thymidylate synthetase:FdUMP:A/5,W10-methylenetetrahydrofolate complex.5 Similarly acid-soluble FdUMP was not increased
by uridine (Chart 3). This suggests that the enhancement by
uridine of FUra action is directed toward the fluororibonucleotide
pool and not the fluorodeoxyribonucleotide pool.
Effect of Undine on the Levels of PRPP. The increase in the
anabolism of 1.0 MM FUra by 10 MMuridine was not associated
with elevated intracellular PRPP levels. Other investigators have
also shown that uridine does not necessarily increase PRPP
levels. For example, treatment of S-49 cells for 4 h with 5 MM
uridine plus 5 MMerythro-9-(2-hydroxy-3-nonyl)adenine,
an adenosine deaminase inhibitor, does not affect PRPP levels (33).
Treatment of rat liver and spleen slices for 3 h with 10 mw uridine
has no effect on PRPP levels (34). Furthermore, neither 0.04 MM
MTX nor 300 MM PALA affected PRPP levels after 6 h of
incubation. Both 0.04 MM MTX and 300 MM PALA inhibited the
growth of S-49 cells by about 90% after 72 h of incubation (data
TIME (hours!
Chart 6. Effect of either FUra or FUra plus uridine (URD) on the incorporation of
|3H]deoxyguanosine into DNA. S-49 cells (3 x 105 cells/ml in 5 ml) were incubated
with FUra (1.0 or 2.4 MM), 1.0 i¡uFUra plus 10 (¿M
uridine, or no drug for 2, 6, and
24 h. pHJDeoxyguanosine (1.0 /jCi/ml) was added to two flasks of each treatment
1 h prior to the end of the incubation. At 2, 6, and 24 h two 2.0-ml aliquots from
each treatment were collected, and the incorporation of [3H]deoxyguanosine into
DNA was determined. The radioactivity (dpm) in DNA from each treatment was
divided by the total number of cells in that treatment, and the data were presented
as percentage of control DNA synthesis. Points, mean from three experiments;
bars, SE. *. significantly different from 1.0 vu FUra. P < 0.05; Newman Keuls'
multiple range test.
CANCER
RESEARCH
not shown), which indicated that MTX and PALA, at concentra
tions which inhibited cell growth did not increase PRPP levels. It
is of interest that Ullman and Kirsch (14) observed that 250 MM
inosine decreases PRPP levels 50% in S-49 cells, which coin
cides with a decrease in the anabolism of FUra and protection
from growth inhibition by FUra. The results of Ullman and Kirsch
suggest a positive correlation between PRPP levels and FUra
anabolism. However, the data reported herein indicated that 10
MMuridine increased both the anabolism of 1.0 MM FUra and its
cytotoxicity, although PRPP levels were not simultaneously in
creased.
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1985
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URIDINE ENHANCEMENT
Effect of Undine on de Novo Pyrimidine Biosynthesis. Un
dine (10 MM) inhibited de novo pyrimidine biosynthesis in S-49
cells by 70% (Chart 5). This inhibiton probably accounted for the
increase in FUra anabolism by undine. Uridine also inhibits de
novo pyrimidine biosynthesis in other systems. For example,
incubation of rat liver and spleen slices with 10 mw uridine for 3
h inhibits de novo pyrimidine biosynthesis by 75%, while there
were no increases in PRPP levels (34). Furthermore, de novo
pyrimidine biosynthesis in rat spleen slices was inhibited only
20% with 1.0 HIM uridine. The apparently high concentration of
uridine (i.e., 10 mw) needed to inhibit de novo pyrimidine biosyn
thesis in this system may be due to the rapid degradation of
uridine by liver or spleen. Tsuboi ef al. (35) found that treatment
of HT-29 cells with 10 UM uridine for 2 h inhibits de novo
pyrimidine biosynthesis by 70%. Similarly, treatment of L1210
cells with 10 MM uridine for 1 h inhibits de novo pyrimidine
biosynthesis by 70% (27).
PALA and uridine, both of which inhibit de novo pyrimidine
biosynthesis, had little effect on PRPP levels (33, 34, 36). To our
knowledge, the only report of increases in PRPP due to PALA is
in MCF-7 cells (37). Therefore, in many tumor cell lines, inhibition
de novo pyrimidine biosynthesis does not necessarily coincide
with increased intracellular PRPP. Our results suggest that an
inhibition of de novo pyrimidine biosynthesis due to uridine,
without any corresponding accumulation of PRPP, is still suffi
cient to allow for increased anabolism of FUra by OPRTase.
Effect of Uridine on UTP Levels. In S-49 cells treated for 6 h
with 10 MMuridine there was a trend (which was not statistically
significant) toward increased UTP levels. Nevertheless, it is prob
able that the levels of UTP which were seen with 10 MMuridine
accounted for the inhibition (via carbamoyl phosphate synthetase
II) of de novo pyrimidine biosynthesis. In L1210 cells a 50%
expansion of the uridine nucleotide pool inhibits de novo pyrimi
dine biosynthesis by 50% (27). By comparison, an apparent
increase of UTP levels in S-49 cells of 46% (which, however,
was not statistically significant; Table 1), was associated with a
70% inhibition of de novo pyrimidine biosynthesis (Chart 5).
Furthermore, the increase in UTP seen in cells treated with 10
MMuridine appeared to compete with FUTP for RNA polymerase,
since the incorporation of FUra into RNA was more closely
related to the FUTP: UTP ratio than to FUTP levels (Table 1). It is
of interest that the combination of PALA plus FUra, which
increases the FUTP:UTP ratio by decreasing UTP levels, also
increases FUra incorporation into RNA as compared to FUra
alone (38).
Effect of Uridine on Macromolecular Synthesis. Uridine (10
MM)did not increase the amount of FdUMP which was bound to
thymidylate synthetase. In addition, free thymidylate synthetase
was apparently still present in cells treated with 1.0 MM FUra,
since treatment with 1.7 MMFUra resulted in increased amounts
of the thymidylate synthetase:FdUMP:A/5,A/10-methylenetetrahydrofolate complex (Chart 4). This indicated that uridine did not
increase the inhibition of thymidylate synthetase by FUra. The
incorporation of [3H]deoxyguanosine into DMA, which was used
as a measure of DNA synthesis (Chart 6), is also an indirect
measure of thymidylate synthetase activity. Incubation of S-49
cells with either 1.0 MM FUra plus 10 MMuridine or 1.0 MM FUra
alone did not affect DNA synthesis even after 24 h. In contrast,
in cells treated with 2.4 MM FUra, DNA synthesis was inhibited
by 50% at 24 h. The differential effect on the inhibition of DNA
CANCER
RESEARCH
VOL.
OF FUra ANABOLISM
synthesis at 24 h by 1.0 MMFUra plus 10 MMuridine as compared
to 2.4 MM FUra suggested that there may be differences in the
mechanism of cell kill due to the two treatments.
Despite the increase in FUra ribonucleotide formation by uri
dine, the enhanced cell kill due to the combination of uridine plus
FUra could not be completely accounted for by increased FUra
anabolism. At most it could account for approximately 50% of
the increased cytotoxicity of FUra plus uridine. Furthermore,
uridine did not appear to increase the inhibition of DNA synthesis
by FUra. In studies to be reported elsewhere6 we have observed
a thymidine-preventable component of the cytotoxicity of 1.0 MM
FUra plus 10 MMuridine which can also account for approximately
50% of the cytotoxicity of this combination. These studies,6
together with the data presented here, indicate that uridine
enhanced FUra cytotoxicity by increasing both the anabolism of
FUra to ribonucleotides and a DNA-directed component of FUra
action.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.
Enhancement by Uridine of the Anabolism of 5-Fluorouracil in
Mouse T-Lymphoma (S-49) Cells
William B. Parker and Philip Klubes
Cancer Res 1985;45:4249-4256.
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