Download Growth Inhibition of a Human Tumor Cell Strain

Growth Inhibition of a Human Tumor Cell Strain by
5-Fluoro-2'-Deoxyuridine. Time Parameters for
Subsequent Reversal by Thymidine*
MAXWELL L . EIDINOFF AND MARVIN A . RICH
(Division of Biophysics, Sloan-KetteringInstitute, Memorial Center, New York, N.Y.)
Experiments related to the inhibition of growth
of H.Ep. #1 cells in culture by 5-fluoro-2'-deoxyuridine (FUDR) and reversal of growth inhibition
by thymidine have been reported by this laboratory (17). These results supported the hypothesis
that a block in the pathway leading to DNAthymine (probably at the "methylation step")
was the principal cause of growth inhibition by
F U D R in this system. This conclusion is in accord
with the results reported by Heidelberger et al.
and in in vitro incorporation studies from this
laboratory with tissue slices (~, 6, 9, 10, 11).
In this report the reversibility by thymidine of
growth inhibition has been studied as a function
of time in which the H.Ep. #1 cells are incubated
in medium containing the FUDR. In addition,
observations concerning the effects on mitotic
index and D N A per cell have been noted.
MATERIALS AND METHODS
Cultures of H.Ep. #1 cells (14), an epitheliallike cell derived from a human cervical carcinoma,
were maintained on Eagle's medium (8) containing
10 per cent horse serum (normal medium). Fourto 6-day cultures were trypsinized (16) by continuous gentle agitation with 0.05 per cent trypsin
Difco (1:~50) at 37~ C. for 5 minutes; 65 X 103
ceils as determined by replicate hemocytometer
counts were added to 60-mm. petri plates containing 4 ml. of the above medium. After incubation
for g4 hours (cells adhere to glass) the medium
was aseptically removed by aspiration, and 4
ml. of fresh medium containing the appropriate
compounds was added to each plate. The plates
were incubated in a humidified chamber at 38 ~ C.
in an atmosphere of carbon dioxide adjusted so
as to maintain the medium at pH 7.6.
* These studies were aided by research grants from the
National Institutes of Health (CY 38~8 and C 8811) and the
U.S. Atomic Energy Commission (AT (80-1)910).
Received for publication December ~2, 1958.
F U D R was synthesized and purified by Hoffmann-La Roche, Inc. (7). Thymidine was purchased from the California Foundation for Biochemical Research.
Stained preparations.--Flates were incubated
in warm saline for g0 minutes, fixed in methyl
alcohol for 30 minutes, and stained with MayGreenwald-Giemsa.
Growth determination.--Cellular growth as measured by total protein was determined after triple
washing to remove culture medium and nonadhering cells, by the colorimetric method of Lowry
et al. (13), as modified by Oyama and Eagle (15).
Microscopic observations.--Frequency of metaphase nuclei was determined on Feulgen stain
preparations at a magnification of 930 X.
D N A determination.--Cells grown on the surface of large Blake bottles were harvested by
trypsinization at various intervals after incubation
in medium containing 4 X 10-TM F U D R . The resultant cell suspensions were washed, the cell
number was determined by replicate hemocytometer counts, and the D N A in replicate cultures
determined by the method of Ceriotti with the
indole-HC1 reaction (8). Highly polymerized D N A
(Worthington Co.) was used as a standard for
the colorimetric assays.
RESULTS
Cell growth following removal of F UDR and
incubation in thymidine-supplemented medium.--In
order to determine the time parameters which
govern the reversal by thymidine of growth inhibition by F U D R , cells were exposed to 4 X
10-7 M F U D R (0.1 ~g/ml) for varying periods of
time (Chart 1). Following removal of the antimetabolite, one replicate group received normal
medium. Thymidine at a concentration of 4 X
10-6 M (1 #g/ml) supplemented the medium of
the other group. Both groups were incubated
for 7 days (with renewal of medium on the 4th
day) at which time cell growth was determined.
5~1
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5~
Cancer Research
Cells exposed to F U D R for as little as 89hour
were unable to multiply when incubated in normal
medium following removal of the F U D R (lower
curves, Chart 1). Cells which were incubated in
F U D R for up to 24 hours were capable of complete
growth when incubated in thymidine-supplemented
medium following F U D R removal (upper curves).
Cell growth measurements were expressed relative
to control plates containing normal medium (no
FUDR).
In the experiments shown in Chart 1, growth of
cells previously treated with F U D R was achieved
by incubation for the entire 7-day period in the
presence of thymidine. To determine whether the
presence of thymidine in the medium for this
period was necessary, the following experiment
was carried out: cells were exposed to 4 X 10-TM
F U D R in normal medium for 19 hours, at which
time the inhibitor was removed and replaced with
Vol. 19, J u n e , 1959
Mitotic activity and cell appearance following
incubation in medium containing F U D R . - - I n
the experiment described in Chart 2, normal medium was replaced by medium containing 4 X
1 0 - 7 M F U D R at zero time. After 24 hours, this
medium was removed and replaced by normal
TABLE 1
EFFECT OF DURATION OF INCUBATION IN
THYMIDINE-SUPPLEMENTED MEDIUI~[ ON
GROWTH FOLLOWING INHIBITION BY
FUDR*
Incubation
time in
normal
medium plus
thymidlne
(hours)
0
2
6
24
52
144
\
80I,-
Z6o_
\~
--EXP.,
"
---EXP.
z
9 FUDR removed
a. 4 0 -_
\
~.,..
FUDR
~"",,....
3 -
2'o-'
medium
(hours)
144
14~
138
1~0
95
0
(per cent)
0
~0+6
13+10
100 + 6
11s
100+9
""......,
20 -
-lb'
Percentage
growtht
* Incubation in normal medium plus F U D R
(4 X 10-7 M) for 19 hours.
t Mean of three replicate cultures _ av. dev.
\ o,,.
Thymidine odded
Incubation
time in
normal
3'o
'
FUDR REMOVED
THYM|DINE ADDEO
'%
I
EXPOSURE TIME TO FUDR (HOURS)
CHART 1.--Effect of incubation time in 4 X 10-7M FUDR
on subsequent growth of H, Ep. #1 cells. Ordinates refer to
percentage growth after incubation for 7 days following exposure to FUDR. The filled circles refer to incubation in normal medium. The two upper curves refer to incubation in
medium supplemented with 4 X 10-~ thymidine.
medium containing 4 X 10-~ M thymidine. At various time intervals the thymidine-containing medium was removed and incubation continued in
normal medium. For each series the total incubation time, i. e., incubation time in normal medium plus thymidine and incubation time
in normal medium, was equal (6 days). Growth
was determined at the end of this period.
From the data in Table 1 it was evident that
thymidine need not be present during the entire
growth period. Twenty-four hours in thymidine
was sufficient to allow complete growth in unsupplemented medium.
After incubation in medium containing F U D R
for 24 hours, there was observed (Chart 1) a
loss in viability which increased with increasing
exposure time to F U D R until very little growth
was observed at 50 hours' exposure.
>~
I
,
I
THYMIDINE
>
~\
/~',
Y\\
I\
TIME (HOURS)
CHART ~.--The effect of F U D R and subsequent treatment
with thymidine on metaphase frequency in H.Ep. #1 cells.
Metaphase frequency represents the percentage of cells in
metaphase at a given time in relation to the percentage just
prior to the addition of FUDR. The latter is based on analysis
of approximately ~500 cells.
medium supplemented with 4 X 10-~ M thymidine,
and incubation was continued for another 24
hours. Replicate plates were removed at various
time intervals during this 48-hour period for mitotic analysis. Although the other mitotic phases
were observed, metaphase frequencies are plotted
in Chart ~ because this stage of mitosis was
considered most favorable for objective scoring.
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EIDINOFF AND R i c H - - T h y m i d i n e Reversal of Growth Inhibition by F U D R
The curves in Chart 2 represent two separate
experiments in which approximately 1000 cells
in replicate plates were examined at each time
interval.
The addition of FUDR was accompanied by
an abrupt cessation of mitosis as evidenced by
the complete absence of metaphase nuclei. Three
hours after the addition, the metaphase frequency
dropped from the control value of 2 per cent
to 0. Small increases appeared sporadically from
15 to 26 hours. At 26 hours (2 hours after the
addition of the thymidine-containing medium)
the incidence of metaphase nuclei began to rise
sharply, and at 36 hours it was more than twice
that of the untreated controls. Following this
peak, the metaphase frequency decreased.
The appearance of the cells following incubation
in normal medium plus FUDR for 72 hours was
observed in plates stained with May-GreenwaldGiemsa. Cells with cytoplasmic projections were
observed after about ~4 hours. The proportion
of bizarre cells increased up to the termination
of this experiment. The nuclear size remained
approximately constant throughout this period.
D N A per cell following incubation of cells in
FUDR-containing medium.--The data presented
in Table 2 indicated that the average DNA content per cell remained relatively constant during
the 24-hour period of incubation in normal medium plus FUDR.
DISCUSSION
The previously reported observation that
growth inhibition by FUDR (4 X 10-7 M) of H.Ep.
#1 cells in culture can be completely reversed
in a noncompetitive manner by thymidine strongly
suggests that the principal site of action of FUDR
(or its metabolic derivative) under these conditions
is at the "methylation step" leading to the thymine moiety (17). The inability of H.Ep. #1
cells to grow out in normal medium after exposure
to FUDR for as little as 89hour suggests a rapid,
irreversible interference by the appropriate metabolic derivative of FUDR with the enzyme sites
involved. This hypothesis is in accord with the
recent demonstration by Cohen et al. that 5-fluorouracil-2'-deoxyriboside-5'-phosphate (FUDRP)
is a highly potent, irreversible inhibitor of a bacterial thymidylate synthetase (5).
The results in Table 1 show that thymidine
is a growth requirement for approximately one
division time following replacement of the FUDRcontaining medium. The noncompetitive reversal
of the FUDR growth inhibition by thymidine,
together with the experiments on labeled orotic
acid and thymidine (17), demonstrated that the
5s
pre-formed thymine moiety is utilized to a greater
extent for DNA synthesis when the de novo synthesis of the thymine moiety is blocked. After
approximately one division time in thymidinesupplemented medium, the newly formed cells
contain a sufficient complement of enzyme sites
free of the active metabolic derivative of FUDR
to permit the normal de novo synthesis of thymidine-5-phosphate. Consequently, there is no further need for thymidine supplement to the normal
medium.
The loss of mitotic activity, together with the
constancy of DNA per cell following incubation
in medium containing FUDR, indicates that DNA
synthesis is significantly suppressed during this
period. These results would be expected to follow
the inactivation of enzyme sites leading to the
de novo thymine moiety (5).
TABLE
THE EFFECT OF F U D R ON
D N A CONTENT PER CELL
Time
DNA/cell
(hours)
(gm. X 1012 __
av. dev.)
--11
--3
O*
+7
+16
-k~o
~o.8_+8%
~1.s_+4%
~1.1 +6%
~4.5_+6%
~.0+5%
$~.9-}-3%
21.7_+5%
* A t zero time F U D R ( 4 X
10 -7 M) was added to the normal
cell medium.
Ackermann et al. (1) have reported the absence
of DNA synthesis in HeLa cells following addition
of 5-fluorouracil to the normal growth medium.
These observations are similar to those reported
above for F U D R and H.Ep. #1 cells. However,
the reversal studies previously reported (17) demonstrate substantial differences in the mechanism
of growth inhibition of H.Ep. #1 cells by 5fluorouracil and FUDR.
The partially synchronous division following
the addition of thymidine to cells that had been
inhibited by FUDR (Chart 2) was not studied
further.
Cohen and Barner have described in bacterial
systems lethal consequences of thymine deficiency
and an accompanying syndrome of unbalanced
growth (4). More recently, Cohen et al. demonstrated that FUDR induces thymine-less death
in E. coli (5). It would be of interest to determine
whether similar phenomena may be observed in a
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524
Cancer Research
mammalian cell system. Ackermann et al. have reported unbalanced cellular growth following maintenance of HeLa cells in 0.5/~g/ml of 5-fluorouracil
(1). Cytochemical studies by Lindner of Ehrlich
ascites tumor cells in mice treated with 5-fluorouracil suggest a similar phenomenon (12). The
results reported here and in the previous paper
(17) on H.Ep. #1 cells and FUDR present several
facets related to the unbalanced growth concept:
(a) H.Ep. #1 cells maintained in FUDR constitute
a thymidine-requiring system; (b) depression of
mitotic activity and constancy of DNA per cell
over the 24-hour period studied; (c) the sharply
reduced cell viability following exposure to FUDR
for longer than 24 hours--this period corresponding to approximately one division time.
This relationship will be more precisely defined
by further studies involving measurement of net
synthesis and turnover of DNA, RNA, and protein during the first 24 hours following incubation
of the cells in medium containing FUDR.
SUMMARY
H.Ep. #1 cells (a human tumor cell strain) required a thymidine supplement to normal growth
medium (Eagle's medium plus serum) after incubation in medium containing 4 X 10-7 M5-fluoro2'-deoxyuridine (FUDR) for as little as 89hour.
Following addition of FUDR to the culture medium, the mitotic index fell significantly, while
the deoxyribonucleic acid per cell remained constant.
These results, with the use of mammalian cell
cultures, are in accord with the recent demonstration by Cohen et al. (5) that 5-fluorouracil-2'
deoxyriboside-5'-phosphate (FUDRP) irreversibly
inhibits bacterial thymidylate synthetase.
When the period of incubation in medium plus
FUDR was increased beyond approximately 24
hours, the cells lost their ability to multiply even
in thymidine-supplemented medium. These results
in thymidine-deficient mammalian cells suggest
a syndrome related to unbalanced growth (4).
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, Hoffmann-La Roche, Inc., kindly supplied samples
of FUDR. The authors are indebted to Mrs. Miriam Black
Vol. 19, June, 1959
for the mitotic observations; Mr. Leonard Saslaw for the
nucleic acid analyses; and Mrs. ganice Bolaffi and Mr. Stanley
Wolf for their assistance in cell cultivation studies.
REFERENCES
1. ACKERMANN,W. W.; Loll, P. C.; and PAYNE, F. E. Effects
of 5-Fluorouracil upon Nucleic Acid Synthesis of Ordinary
and Viral Infected HeLa Cells. Bact. Proc., p. 71, 1958.
2. BOSCH, L.; HARBERS, E.; and HEIDELBERGER,C. Studies
on Fluorinated Pyrimidines. V. Effects on Nucleic Acid
Metabolism in Vitro. Cancer Research, 18:385-48, 1958.
3. CEPJOTTI, G. Determination of Nucleic Acids in Animal
Tissues. J. Biol Chem, 214:59-70, 1955.
4 COHEN, S S , and BARNE~ H. D. Studies on Unbalanced
Growth in E. coll. Proc. Nat. Acad. Sc., 40:885-98, 1954.
5. COHEN, S. S.; FLAKS, J. G.; BARNER, H. D.; LOEB
M. R.; and LICHTENSTEIN, J. The Mode of Action of
5-Fluorouracil and Its Derivatives. Proc. Nat. Acad. So.,
44:1004-12, 1958.
6. DANNEBERG, P. B.; MONTAG,B. J.; and HEIDELBERGER,
C. Studies on Fluorinated Pyrimidines. IV. Effects on
Nucleic Acid Metabolism in Vivo. Cancer Research, 18:
829-84, 1958.
7. DUSCHINSKY,R.; PLEVEN, E.; MALBICA,J.; and HEIDELBERGER,C. Synthesis of 5-Fluorouracil Nucleosides. Abstr.,
132d Meeting, Am. Chem. Soc., p. 19C, 1957.
8. EAGLE, H. Nutrition Needs of Mammalian Cells in Tissue
Culture. Science, 122: 501-4, 1955.
9. EmINOFF, M. L.; KNOLL, J. E.; and KLZIN, D. Effect
of 5-Fluorouracil on the Incorporation of Precursors into
Nucleic Acid Pyrimidines. Arch. Biochem. & Biophys.,
71: 274-75, 1957.
10. HEIDELBERGER, C.; BoscH, L.; CHAUDHURI, N. K ; and
DANNEBEaG, P. B. Mechanism of Action of 5-FluoroPyrimidines. Fed. Proc., 16:194, 1957.
11. HEIDELBERGER, C.; CHAUDHURI,N. K.; DANNEBERG, P.;
MOOREN, D.; GRIESBACH,L.; DUSCHINSKY, R.; SCHNITZER, R. J.; PLEVEN, E.; and SCHEINER, J. Fluorinated
Pyrimidines, a New Class of Tumor Inhibitory Compounds. Nature, 179: 663-66, 1957.
12. LINDNER, A. Cytochemica] Effects of 5-Fluorouracil on
the Deoxyribonucleic Acid and Protein of Ehrlich Ascites
Tumor Cells. Proc. Am. Assoc. Cancer Research, 2:322,
1958.
13. LowRY, O. H.; ROSEBROUGH,N. J.; FAIR, A. L.; and RanDALL, R. J. Protein Measurement with the Folin Phenol
Reagent. J. Biol. Chem., 193:265-75, 1951.
14. MOORE, A. E.; SABACHI~-WSKY,L.; and TOOLAN, H. W.
Culture Characteristics of Four Permanent Lines of Human Cancer Cells. Cancer Research, 15: 598-602, 1955.
15. OYAMA,V. I., and EAGLE, H. Measurement of Cell Growth
in Tissue Culture with a Phenol Reagent (Folin-Ciocalteau). Proc. Soc. Exper. Biol. & Med., 91:305-7, 1956.
16. PUCK, T. T.; MARcus, P. I.; and CIECIURA, S. J. Clonal
Growth of Mammalian Cells in Vitro. Growth Characteristics of Colonies from Single HeLa Cells with and without a "Feeder" Layer. J. Exper. Med., 103:273-84, 1956.
17. I~CH, M. A.; BOLAFFL J. L.; KNOLL, J. E.; CHEONG,
L.; and EIDINOFF, M. L. Growth Inhibition of a Human
Tumor Cell Strain by 5-Fluorouracil, 5-Fluorouridine, and
5-Fluoro-~'-deoxyuridine---Reversal Studies. Cancer Research, 18: 730-35, 1958.
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Research.
Growth Inhibition of a Human Tumor Cell Strain by 5-Fluoro-2′
-Deoxyuridine: Time Parameters for Subsequent Reversal by
Thymidine
Maxwell L. Eidinoff and Marvin A. Rich
Cancer Res 1959;19:521-524.
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