Download Growth Inhibition of a Human Tumor Cell Strain

Growth Inhibition of a Human Tumor Cell Strain by
5-Fluorouraeil, 5-Fluorouridine, and 5-Fluoro-2'deoxyuridine—Reversal Studies*
MARVINA. RICH,JANICEL. BOLAFFI,JOSEPHE. KNOLL,f
LORETTACHEONG,ANDMAXWELLL. EIDINOFF
(Division of Biophysics, Sloan-Kettering Institute, Memorial Center, New York, N.Y.)
The inhibitory effect of 5-fluorouracil (FU),
5-fluorouridine (FUR), and 5-fluoro-2'-deoxyuridine (FUDR) on tumor and microbial growth has
been recently reported by Heidelberger, Duschinsky, and co-workers (4, 9, 11, 12, 23). Tracer
studies demonstrated that these compounds se
lectively depressed the incorporation of labeled
precursors into the thymine moiety of nucleic
acids (6, 9-11). In the experiments reported be
low, the mechanism of growth inhibition by these
compounds was studied with H.Ep. #1 cells grown
in tissue culture.
Differences in the ability of selected compounds
to reverse the growth inhibition by these fluorinated pyrimidines were measured to test the pos
sibility of a single primary site of growth inhibi
tion. The effect of FUDR on the utilization of
isotopically labeled precursors for nucleic acid
pyrimidines of H.Ep. #1 cells in tissue culture
was studied in a medium containing thymidine
as a reverser of growth inhibition.
MATERIALS AND METHODS
5-Fluorouracil, 5-fluorouridine, and 5-fluoro-2'deoxyuridine were synthesized and purified by
the Hoffman-La Roche Co. (4). Thymidine, cal
cium thymidylate, deoxyuridine, uridine, cytidine,
uracil, cytosine, and thymine were purchased from
the California Foundation for Biochemical Re
search. 5-Methyldeoxycytidine was synthesized
by the Synthetic Organic Section, Sloan-Kettering
Institute (7). Thymidine labeled with tritium by
catalytic exchange and having a specific activity
of 320 fiC/foaole was purchased from the Schwarz
* These studies were aided by research grants from the
National Institutes of Heath (CY 3328) and the U.S. Atomic
Energy Commission (AT (30-1) 910).
t Present address: Squibb Institute for Medical Research,
New Brunswick, N.J.
Received for publication February 14, 1958.
Laboratories. Thymidine-2-C14 was prepared from
thymine-2-C14 (Volk Laboratories, Chicago, Illi
nois) and thymidine in the presence of an E. cdi
extract (22) and had a specific activity of 0.50
/ic/jumole. Orotic acid-6-C14 (New England Nu
clear Co.) had a specific activity of 2.5 /iC/jumole.
Tissue culture methods.—Stockcultures of a human cervical
carcinoma, H.Ep. #1 (19), were maintained in bottles on
Eagle's medium containing 10 per cent horse serum (5).
Four- to 6-day stock cultures were trypsinized (21) by con
tinuous, gentle agitation with 0.05 per cent trypsin (Difco
1:250) at 37°C. for 5 minutes. Between 20,000 and 50,000
cells, as determined by replicate hemocytometer counts, were
added in triplicate to 60-mm. petri plates containing 4 ml.
of the above medium. After incubation for 24 hours, the
appropriate concentrations of inhibitor and reverser (sterilized
by passage through Millipore filters) were added and the plates
incubated for 7 days. Renewal of medium and addition of
compounds were repeated after 4 days. Incubation was carried
out in a humidified chamber at 38°C. in an atmosphere
of carbon dioxide adjusted so as to maintain the medium
at pH 7.6. Growth measured as total protein was determined
(after triple washing to remove medium and nonadhering
cells) by the colorimetrie method of Lowry et al. (16), as
modified by Oyama and Eagle (20). To determine the degree
of inhibition and subsequent reversal, growth in the control
plates (no additions) at 7 days was taken as 100 per cent, with
protein values at 24 hours representing zero growth. Control
plates exhibited a 6- to 9-fold increase in total protein during
the 7-day incubation period.
DNA determinations.—The experiments summarized in
Table 3A were designed to measure the uptake of tritium labeled thymidine into DNA of growing cells; IO4 to IO5
cells were added to 10 ml. of the above medium in 100-ml.
dilution bottles (surface area 44 sq. cm.). The medium was
replenished, and the compounds were added after 24 and
96 hours. The experiments were terminated after 7 days, at
which time the bottles were rinsed twice with saline. The
DNA determinations were carried out by the method of McIntire and Sproull, with precipitation of DNA by salmine
and solution of the purines and pyrimidines by hot acid-salt
solution (18). Aliquota containing approximately 50 ng. of
the DNA-salmine precipitate were transferred to aluminum
planchéis(20 sq. cm.) and counted in a windowless gas-flow
counter. The specific activities obtained in this manner are
listed in Table 3A. The DNA-salmine precipitate could be
dissolved in 0.4 N NaOH and repiecipitated without a signifi
cant change in the amount and specific activity.
730
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RICH et al.—Inhibition of H.Ep. fi Cells by 5-Fluorouracil
Spécifia
activity of nucleic acid pyrimidines.—After decanting
of the culture medium, the cells were washed twice with isotonic
saline, 3 times with 2 ml. cold 0.4 N perchloric acid (PCA),
and successively with 75 per cent ethanol, 95 per cent ethanol,
absolute alcohol, 3:1 ethanol-ether at 40°C. (twice), and ether.
The residue (weighing about 10 mg.) was incubated with
0.5 ml. of 0.3 N KOH for 18 hours at 37°C., and the DNA
and protein were precipitated by cold PCA as described
by Davidson and Smellie (3), washed with cold 0.4 N PCA
3 times, with alcohol, and then dried in a nitrogen stream.
The supernatant containing the ribonucleotides was also evap
orated to dryness. The free bases were obtained by the method
of Marshak and Vogel (17). The individual bases were sepa
rated by two-dimensional paper chromatography (14). After
elution of the pyrimidine bases from the paper (Whatman
#1), the specific activity was determined. The C14 activity
was based on gas-flow counter measurements with the use
of 20-sq. cm. aluminum planchéis.The concentration was
731
(Chart 1) for FUR and FUDR. However, the
nucleosides were effective inhibitors at approxi
mately one-hundredth
the concentration
of FU.
Each point in Chart 1 is the average of three
replicate cultures.
Attempts to reverse growth inhibition by FU,
FUR, FUDR.—Several compounds related to me
tabolites on pathways
leading to nucleic acid
pyrimidines were tested for their ability to reverse
growth inhibition by FU, FUR, and FUDR. Uracil, cytosine, and thymine did not reverse growth
inhibition by FU, FUR, and FUDR even at
a concentration
100 times greater than those of
the fluorinated compounds. In Table 1, a reversal
TABLE 1
CONCENTRATION OF 5-FLUOROURACIL-MICROGRAMS/ML.
O.I
1.0
10.0
EFFECTOFSEVERAL
COMPOUNDS
ONGROWTH
INHI
BITIONOFH.EP.#1CELLSBYFUR ANDFUDR
RATIOor
CONCENTRA
100-
»40
0
TION«:
COMPOUND
Thymidine
eon
Thymidylate
(calcium)
LEGEND
+ 5-FLUOROURACIL (UPPER SCALE)
•5-FLUOROURIDINE
O 5-FLUORO-2-DEOXYURIDINE
20-
5-methyldeoxycytidine
Deoxyuridine
0.001
0.01
CONCENTRATION
OF 5-FLUOROURIDINE
AND
5-FLUORO-2-DEOXYURIDINE- MICROGRAMS
/ML.
OJ
CHART1.—Growth inhibition of H.Ep. #1 cells by 5-fluorouracil (FU), 5-fluorouridine (FUR), and 5-fluoro-2'-deoxyuridine (FUDR).
calculated from optical densities at the absorption maxima
(pH 2) with an appropriate blank based on elution of a control
strip of paper. The purity of each pyrimidine was checked
by the measurement of optical density ratios at different
wave lengths. Preliminary experiments demonstrated that
the adenine and guanine had less than 1 per cent of the
specific activity of the pyrimidines.
When thymidine-C14 was used as precursor, the cells were
digested with 0.4 N NaOH as described above (18). The RNA
was hydrolyzed to ribonucleotides in this manner, and the
DNA plus protein was precipitated with 70 per cent PCA.
Only DNA thymine was isolated. This procedure yielded
the results listed in Table 3B.
RESULTS
Inhibition by FU, FUR, FUDR—It is evident
from Chart 1 that FU, FUR, and FUDR are
potent inhibitors of H.Ep. #1 cells. Essentially
complete growth inhibition was observed at 1.0,
0.01, and 0.01 Mg/ml of FU, FUR, and FUDR
(80, 0.4, and 0.4 X IO-'M), respectively.
There
was relatively
little difference in the toxicity data
Uridine
CoiipocND
INHIBITOB
1
10
100
1
10
100
1
10
100
REVERSALor OBOWTHINHIBITION
FUR (4X10-'«)
FUDR (4X10-'M)
—
—
—
-
+*
+
+
+
+
—
+
+
1
10
100
1
10
100
Cytidine
1
10
100
* Tests are referred to as + when the cell growth reached
30 per cent of the growth in the control cultures.
test is scored minus or plus if the net protein in
crease after 7 days is less than or greater than
30 per cent, respectively, of the net protein in
crease in the control cultures. The four compounds
that reversed the growth inhibition by FUDR
are, in order of decreasing activity: thymidine,
thymidylate,
5-methyldeoxycytidine,
and deoxyuridine. These four deoxyribose-containing
com
pounds did not reverse the growth inhibition by
FUR, while the ribosides uridine and cytidine
did reverse at levels 100 times the concentration
of FUR. The compounds in Table 1 did not reverse
the growth inhibition by FU.
Since the concentration
of deoxyuridine
re
quired to reverse FUDR inhibition was 100 times
the concentration
of thymidine necessary to ac-
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732
Voi. 18, July, 1958
Cancer Research
complish reversal, it seemed desirable to compare
the two systems with respect to the type of re
versal involved. Accordingly, a series of concen
trations of each compound was tested against
two concentration levels of FUDR.
The two upper curves in Chart 2 represent
the reversal of FUDR growth inhibition by thymidine at inhibitor levels differing by a factor
of five. Their similarity indicates that, over this
FUDR range, the reversal depends simply on
the concentration of thymidine in the medium
and is not dependent on the reverser-inhibitor
ratio. The two lower curves represent reversals
by deoxyuridine. A fivefold increase in the FUDR
level required an approximately fivefold increase
THYMIDWE-MICROGRAMS/ML.
Dß THYMIDINE (UPPER SCALE)
O« DEOXYURIDINE I LOWER SCALE)
RNA-pyrimidines was not appreciably affected
by the presence of FUDR. In the absence of
the exogenous thymidine, this concentration of
FUDR would have caused complete growth in
hibition.
The tracer studies with the use of precursor
thymidine are summarized in Table 3. In Part
A, thymidine-H3 was used in four experiments
over a period of about 1 month. The results in
the last column express the increased specific
activity of the DNA formed in the presence of
FUDR and thymidine as compared with the nor
mal medium augmented only by thymidine. Since
the precursor is incorporated predominantly into
the DNA-thymine, these results reflect the ratios
of the DNA-thymine specific activities. In Part
B, thymidine-C14 was used and the DNA-thymine
measured directly. The average deviations listed
in Table 3 indicate significant biological variations
in the replicate cultures used. The results of all
these experiments demonstrate a two- to fourfold
TABLE 2
EFFECTOFFUDR ONTHEINCORPORATION
OFOROTIC
ACIDINTOTHE NUCLEICACIDPYRIMIDINES
OFH.Ep. #1 CELLS
Specific activity
counts/nrin/>imoleX 10"'
~T
40
60
OEOXYURIDINE-MICROGRAMS/ML
CHART2.—Effect of thymidine and deoxyuridine on the
growth of H.Ep. #1 cells in the presence of two concentrations
of 5-fluoro-2'-deoxyuridine (FUDR).
in the deoxyuridine level to maintain the same
percentage of growth (relative to control cultures).
This calculation was made using the ordinate
at 40 per cent of control growth. The lower
curves could not be carried to 100 per cent of
control growth, since deoxyuridine at sufficiently
high concentrations becomes growth-inhibitory
under these conditions. The results of a second
study of the reversal action of thymidine and
deoxyuridine yielded results essentially equal to
those plotted in Chart 2.
Tracer studies.—The specific activities of the
nucleic acid pyrimidines of H.Ep. #1 cells grown
in a medium containing thymidine in the presence
and absence of FUDR and with orotic acid-C14
used as precursor are listed in Table 2. In the
presence of FUDR, the DNA-thymine specific
activity was depressed in two experiments by
a factor of 4.9 (Exp. A) and by 5.4 (Exp. B.),
respectively. In the latter experiment, the FUDR
concentration was increased fivefold. The utiliza
tion of the orotic acid for DNA-cytosine and the
A. Thymidine in medium
(Mg/ml)
FUDR in medium
(iig/ml)
DNA Thymine
Cytosine
RNA Uracil
Cytosine
B. Thymidine in medium
(ng/ml)
FUDR in medium
(Mg/ml)
DNA Thymine
Cytosine
RNA Uracil
Cytosine
1
1
0
0.1
4.72
6.25
9.8
6.4
0.97
7.0
9.0
7.6
1
Ratio
4.9
0.9
1.1
0.84
05
5.4
6.25
10.0
7.0
1.00
6.35
14.8
10.3
5.4
1.0
0.68
0.68
increased utilization of exogenous thymidine in
the formation of DNA when the inhibitor is
added to the medium, in the presence of the revers
ing agent.
DISCUSSION
The growth inhibition data in Chart 1 demon
strate in the mammalian cell the increased in
hibitory effect of the riboside or deoxyriboside
as compared with the pyrimidine base. This effect
has been noted by Schindler and Welch in the
case of 6-azauracil and its riboside with Sarcoma
180 cells used in tissue culture (24). The inhibition
data for FUR and FUDR are roughly equivalent.
The reversal studies discussed below indicate dif
ferences in mechanisms of growth inhibition.
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RICH et al.—Inhibition
of II.Ep.
The growth inhibition caused by FUDR (0.1
and 0.5 ¿ig/ml) was completely prevented by
approximately 0.5 fig of thymidine/ml (Chart
2). These results suggest that the FUDR is block
ing the formation of metabolites containing the
thymine moiety and leading to DNA thymine.
The decreased incorporation of orotic acid-C14
into DNA-thymine in the presence of FUDR
(Table 3) demonstrates a specific interference with
this pyrimidine ¿moiety,since the other nucleic
acid pyrimidines are affected only to a slight
extent. This site of action of the fluorinated py
rimidines has been reported by Heidelberger et al.
and by this laboratory (6, 9-11) for experiments
in which uptake of the labeled precursor was
fi Cells by 5-Fluorouracil
733
1), together with its dependence on the FUDR
concentration (Chart 2), suggests a competitive
mechanism at a level leading to the formation
of the thymine-moiety. The studies of Friedkin
and Kornberg have indicated that this may be
at the level of deoxyuridine-5-phosphate (8).
Heidelberger et al. have suggested that the
primary metabolic block for FU, FUR, and FUDR
may involve this methylation step (10). However,
the inability of thymidine to reverse the growth
inhibition by FU and FUR (Table 1), even at
a concentration 100-fold in excess of that required
to reverse the same level of FUDR inhibition,
strongly suggests that FU and FUR are producing
metabolic blocks essential to growth, in addition
TABLE 3
EFFECTOFFUDR ONTHEINCORPORATION
OFTHYMIDINEINTOTHE
DNA OFH.EP. #1 CELLS
A. PRECURSOR:THTumniE-H'
Eip.
no.
1
2
3
4
Activity
in medium
Days of
incubation
with labeled
precursor
0.1
0.01
0.01
0.1
6
8
5
1
DNA (counts/min/Vg)
Thymidine*
Thymidine
alone
+FUDH
1530±150f(2)î
30 ± 7 (3)
168± 5(2)
100± 16(3)
417±33
±33 (2)
13+
+ 0.3(3)
S6± 8 (3)
31 + 13 (3)
B. PRECURSOR:
THYMIDINE-C"
DNA-thymine specificactivity
(counts/min//<mole X10-')
Thymidine
Thymidine
+FUDH
alone
Relative
activity
3.7§
2.3
3.0
3.2
Relative
activity
0.002
6
15.0±3 (2)
7.2± 1.2(2)
2.1
* FUDR, 0.1 /ig/ml; thymidine, 1 tig/mi.
t Av. deviation for replicate cultures.
i No. of replicate cultures.
§Ratio of averages in preceding columns.
studied over a relatively short period. The results
presented in Tables 2 and 3, however, were ob
tained during a several-fold increase in total DNA.
The increased incorporation of precursor thymi
dine into DNA-thymine in the presence of FUDR
demonstrates that at least part of the reversal
mechanism involves direct utilization of the ex
ogenous thymidine for DNA-thymine.
The reduced effectiveness of thymidylate as
compared with thymidine (Table 1) may indicate
the necessity for dephosphorylation prior to utili
zation by the cell for incorporation into DNA
thymine (15). The reversal by 5-methyldeoxycytidine suggests that this compound may be
utilized in pathways leading to DNA-thymine in
mammalian cells. Cohen and Barner (2) have
recently shown that a thymine-requiring strain of
E. coli can utilize 5-methyldeoxycytidine to fill its
thymine-requirement and permit normal growth.
The hundred-fold excess of deoxyuridine required
to reverse the growth inhibition by FUDR (Table
to the "methylation block" suggested for FUDR
inhibition. These additional sites probably involve
the formation of F-containing pyrimidine ribonucleotides, nucleotide di- and tri-phosphates,
coenzymes, and the incorporation of the F-uracil
moiety directly into RNA, as demonstrated by
Heidelberger (9, 11). The inability of any of the
compounds in Table 1, as well as the pyrimidine
bases themselves, to reverse the growth inhibition
by FU further suggests the presence of multiple
sites of growth inhibition.
A significant difference between these reversal
studies and the results of Scheiner et al., with
L. leichmanii (23), is the failure of the pyrimidine
bases to reverse growth inhibition by FU, FUR,
and FUDR in H.Ep. #1 cells. These observations
are in accord with the relatively poor utilization
in mammalian cells of the pyrimidine bases, as
compared with their corresponding nucleosides
(1, 13).
The tracer study results described in Tables
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734
Cancer Research
2 and 3 are consistent with the view that the
utilization of the preformed thymine moiety
is enhanced under conditions where the de ñauo
pathway (from orotate) is partially blocked. Simi
lar conclusions were reached from tracer studies
following incubation of tumor tissue slices for
2 hours in Krebs-Ringer solution containing 0.1
per cent glucose (6). The tracer studies carried
out in tissue culture in the presence of inhibitor
plus reverser are of special interest, because the
cells are able to multiply at approximately the
same rate as in the presence of the reverser alone.
The data in Tables 2 and 3 thus refer to utilization
of labeled precursors in the formation of new
nucleic acids. In the short incubation of slices,
breis, and homogenates, the data may reflect
an exchange as well as a net synthesis of nucleic
acids. Consequently, tracer studies in growing
cells in the presence of both inhibitor and reverser
(where a suitable system can be found, such
as that in Tables 2 and 3) can provide significant
information on mechanisms of inhibitor action.
SUMMARY
The effects of 5-fluorouracil (FU), 5-fluorouradine (FUR), and 5-fluoro-2'-deoxyuridine (FUDR).
on the growth of H.Ep. #1 human cell strain
in a semisynthetic medium have been studied.
FU, FUR, and FUDR produced complete inhibi
tion of growth at 1.0, 0.01, and 0.01 ¿tg/ml.,
respectively (8 X IO"6, 0.4 X IO"7, and 0.4 X
10~7M).
The complete growth inhibition by FUDR at
4 X 10~7Mis reversed by an approximately equiva
lent concentration of thymidine. The extent of
reversal is independent of the FUDR concen
tration over a fivefold variation in the latter.
These results strongly support the hypothesis that
a block in the pathway leading to DNA-thymine
(probably at the "methylation step") is the prin
cipal cause of growth inhibition by FUDR. How
ever, the inability of thymidine to reverse the
growth inhibition by FU and FUR suggests that
the latter are producing metabolic blocks at sites
essential to growth in addition to the "methylation
block" suggested for FUDR inhibition.
The growth inhibition by FUDR is reversed
by deoxyuridine at an approximately 100-fold
excess. A fivefold increase in FUDR concentration
increased by a corresponding factor the level of
deoxyuridine needed for reversal. The inhibition
of growth by FU could not be reversed by a
100-fold excess of thymidine, 5-methyldeoxycytidine, deoxyuridine, uridine, cytidine, uracil, thy
mine, and cytosine.
The specific activity of the nucleic acid pyrimi-
Vol. 18, July, 1958
dines of H.Ep. #1 cells grown in the presence of
FUDR plus thymidine (as reverser) was compared
with cells grown in the presence of an equal con
centration of thymidine alone. With labeled orotic
acid, a significant depression in the specific ac
tivity of DNA-thymine was observed in the pres
ence of FUDR, with relatively little effect on
the other nucleic acid pyrimidines. With labeled
thymidine, the specific activity of the DNAthymine was increased by a factor of two to
four in the presence of FUDR. These results
demonstrate an enhanced utilization of exogenous
thymidine under conditions in which the de novo
pathway to DNA-thymine is blocked by FUDR.
ACKNOWLEDGMENTS
The stock culture of H.Ep. #1 cells was kindly given to our
laboratory by Miss L. Diamond and Dr. A. E. Moore, Virus
Study Section, Sloan-Kettering Institute. Dr. L. Duschinsky,
Hoffman-La Roche Co., kindly supplied samples of FU, FUR,
and FUDR. The authors are indebted to Mr. Leonard Saslaw
for valuable assistance in the tracer experiments.
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Growth Inhibition of a Human Tumor Cell Strain by
5-Fluorouracil, 5-Fluorouridine, and 5-Fluoro-2 ′-deoxyuridine−−
Reversal Studies
Marvin A. Rich, Janice L. Bolaffi, Joseph E. Knoll, et al.
Cancer Res 1958;18:730-735.
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