Effect of 5-Bromodeoxyuridine on Deoxyribonucleic AcidThymine Synthesis and Cell Metabolism of
Lymphatic Tissues and Tumors*
SAULKIT, CHARLESBECK,ODETTEL. GRAHAM,ANDARTHURGROSS
(Department of Biochemistry, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston 25, Texas)
5-Bromodeoxyuridine (BrUDr) is an effective
inhibitor of the growth of E. coli and Lactobacitti,
and this growth inhibition can be reversed by
thymidine (1-3). There is evidence that this thymidine analog can replace the thymine of both
E. coli and bacteriophage deoxyribonucleic acid
(DNA) on an equimolar basis (7, 11, 12). In
E. coli, nearly 50 per cent of the thymine can
be replaced. Whereas the physical properties and
appearance of bacteriophage particles containing
incorporated 5-bromouracil appeared normal, a
proportion were found to be nonviable. Litman
and Pardee have observed that T2r2 stocks grown
in the presence of 5-bromouracil and sulfanilamide
under conditions in which the 5-bromouracil is
incorporated into the DNA may contain a high
proportion of mutant types (11). The mutagenic
effect of 5-bromouracil in the presence of sulfanil
amide must certainly be connected with a dis
turbance of DNA metabolism or structure. Anoth
er instance of mutagenesis resulting from an inter
ference with thymine metabolism has been pre
sented by Coughlin and Adelberg (6), who ob
served that specific thymine starvation is muta
genic in an E. coli strain which has a double re
quirement for thymine and histidine. A hundred
fold proportional increase of histidine-independent
mutants appeared among the survivors of 185
minutes of thymine starvation.
These facts prompted a study of the potential
role of BrUDr as an inhibitor of thymine bio
synthesis by tumors. The effect of the analog
on the incorporation of formaldehyde-C14 into
thymine compounds under in vivo or in vitro
conditions was investigated. Inhibition was ob
served in the labeling of DNA-thymine, but the
labeling of DNA-adenine or guanine was reduced
only at high concentrations of BrUDr. Other
analogs of thymidine had no significant effect
* Aided in part by grants from the American Cancer Society
and the Leukemia Society, Inc.
Received for publication January 9, 1958.
under the same conditions. Although the incor
poration of isotopically labeled one-carbon com
pounds into DNA-thymine was inhibited, neither
the labeling of acid-soluble thymine compounds,
serine, nor proteins was inhibited, nor was cell
glycolysis or respiration significantly reduced. It
would therefore appear that BrUDr represents
a relatively specific antagonist of the terminal
steps in the utilization of thymidine for DNA
synthesis.
MATERIALS
AND METHODS
Cell suspensions of the following tissues were used in this
study: rat thymus, mouse spleen, lymphatic leukemia LL5147
(Ak mice) and E9514A (C3H mice), and lymphosarcoma
6C3HED. The care and handling of the tumor-bearing animals
and preparation of the cell suspensions have been previously
described (9, 10).
Cell suspensions (approximately 20 mg. dry weight of tis
sue) were incubated at 88°C. in Warburg vessels containing a
total volume of 2.45 cc. of fluid. The flasks also contained 2.5
/'•
inîles
of glutaminc, 30 /¿molesof glucose, 1 /¿moleof deoxycytidine, in some instances 1 Minole of thymidine, varying
concentrations of BrUDr or other antimetabolites, and the
labeled precursor. The incubation tune was generally 3 hours.
Formaldehyde-C'4 (1 /imole [2 juc] per flask) was obtained
from Isotope Specialties, Inc. 5-Bromodeoxyuridine, 5-hydroxydeoxyuridine, and 5-bromouridine, bromouracil, and
6-mercaptopurine were purchased from the California Founda
tion for Biochemical Research and Nutritional Biochemicals,
Inc. The authors are indebted to Dr. James F. Holland of the
Roswell Park Memorial Institute, Buffalo, New York, for a gift
of 2-methylmercapto-4-amino-5-hydroxymethyIpryimidine.
Acid-soluble thymine, thymidine, and thymidylate and
DNA-thymine were extracted from the tissues, subjected to
purification from radioactive contaminants, and assayed for
radioactivity as previously described (10). Respiration and
glycolysis were measured by conventional manometric proce
dures. The methods for determining the specific activity of free
serine and protein were those previously used in this laboratory
(9). Details concerning the in vivoexperiments are given in the
results section of this paper.
RESULTS
Effects of bromodeoxyuridine on the in vivo in
corporation of formaldehyde-Câ„¢into DNA-thymine.
—C3H mice weighing 20-25 gm. were given in
oculations intraperitoneally of 0.2 cc. of un-
598
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KIT et al.—5-Bromodeoxyuridine
and Cell Metabolism
599
diluted ascites fluid containing E9514A tumor cells. to DNA. BrUDr at levels ranging from 0.32
After 7 days of tumor growth, the mice were to 3.2 milJimoles was effective in reducing the
divided into two groups of three. One group labeling of DNA-thymine. In the experiments
was given injections intraperitoneally of 5.4 /¿moles described above, 1 /¿moleof deoxycytidine was
of BrUDr, the other of physiological saline. Five present in the incubation medium. As reported
minutes later, 4 //.c. (2 /¿moles)
of formaldehyde-CH
previously, the presence of deoxycytidine greatly
was administered by the same route, and 2 hours increases the conversion of the one-carbon pre
afterwards the animals were sacrificed. BrUDr cursor to thymine compounds (10). Inhibition
of DNA-thymine labeling was, however, obtained
markedly inhibited the incorporation of the pre
cursor into the DNA-thymine of the tumor cells whether or not deoxycytidine was included in
or the spleen cells from the tumor-bearing animal the medium (Table 3). Several experiments were
but did not significantly affect the labeling of also performed in which nonradioactive thymidine
DNA-adenine or guanine (Table 1).
was added to the incubation flasks. Thymidine
TABLEl
EFFECTOFS-BROMODEOXYURIDINE
ONINCORPORATION
OF FORMALDEHYDE-C14 INTO DNA In Vivo
Thymine
Control
BrUDr*
TISSUE
E9514A
Spleen
1250
1300
Adenine
Control
BrUDr*
450
580
3500
4710
3270
3940
Guanine
Control
BrUDr*
2090
2260
1210
1490
2580
2040
E9514A
1370
270
* Bromodeoxyuridine
(5.4 /¿moles)injected 5 minutes prior to a test dose of 4 /ic. of
HjC"O (2 Amóles), and animals sacrificed 2 hours later.
TABLE 2
INHIBITIONBY S-BROMODEOXYURIDINE
OF INCORPORATION
OF FORMALDEHYDE-C14 INTO DNA-THYMINE
TISSUE
Gardner
ADDITION
Control
BrUDr
1.6
DNA
TAG
(count«/min//imole)
4930
2800
1970
1390
3040
1860
LL5147
Control
BrUDr
0.8
1985
496
Control
BrUDr
BrUDr
BrUDr
T = thymine;
0.8
1.6
2.4
11920
6180
4480
3610
Thymus
(rat)
Abbreviations:
5-bromodeoxyuridine.
Cose,
(millimole«)
A = adenine;
3410
3190
1840
1595
G = guanine;
In vitro effect of 5-bromodeoxyuridine.—The
presence of BrUDr also resulted in a marked
in vitro inhibition of the incorporation of formaldehyde-C14 into the DNA-thymine of rat thymus,
lymphatic leukemias LL5147 and E9514A, and
lymphosarcoma 6C3HED (Table 2). The specific
activity of DNA-adenine or guanine was not af
fected at low concentrations of BrUDr but was
progressively reduced at higher concentrations.
There was no apparent reduction of the labeling
of acid-soluble thymine, thymidine, or thymidylate. Instead, the total radioactivity of the acidsoluble thymine compounds was increased as DNA
biosynthesis decreased. It is therefore unlikely
that the BrUDr was inhibiting the methylation
of the thymidine precursor. Rather, the effect
was apparently on the conversion of the thymidine
TDr
in Vitro
ACID-SOLUBLE
TDr
TDrP
(total counts/min)
3710
10500
470
3100
8670
360
T
1150
3210
650
1240
4230
1570
580
5010
2225
565
6745
2220
910
8705
2680
1010
5440
1845
thymidine; TDrP = thymidylate; BrUDr =
TABLE 3
In Vitro EFFECTOFS-BROMODEOXYURIDINE
ON THE LA
BELINGOFTHYMINECOMPOUNDS
OFLYMPHOSARCOMA
6C3HED WITHORWITHOUTDEOXYCYTIDINE
ADDED
ADDIATioNS
Control
3710CDr*
5850BrUDr*
DNA
T
(counts/min/pmole)
2480
440CDr*38705260
1970
5070
ACID-SOLUBLE
T
TDr
TDrP
(total counts/min)
1010
660
130
1460
7200
270
1010
1000
80
1640
7480
290
BrUDr
* One /umole of deoxycytidine and 2.17 /amólesof bromodeoxyuridine per flask. Each flask also contained 30 /¿molesglu
cose, and 2.5 jumólesglutamine, and 1 /umole (2 /¿c.)of formaldehyde-C14. Total volume, 2.45 cc.
Abbreviations:
CDr = deoxycytidine;
BrUDr = bromodeoxyuridine.
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Vol. 18, June, 1958
Cancer Research
600
markedly depressed the labeling of DNA-thymine,
but not of DNA-adenine.
Because the size of
the free thymidine pool was increased, the total
radioactivity
of free thymine compounds was in
some cases increased. The labeling of acid-soluble
thymine compounds was investigated in the pres
ence of BrUDr and thymidine but in the absence
of deoxycytidine.
Under these conditions also,
BrUDr did not inhibit the labeling of acid-soluble
thymine compounds (Table 4).
Effect of compounds related to 5-bromodeoxyuridine.—The effect of various compounds related to
BrUDr on the labeling of thymine compounds was
investigated.
At comparable concentrations,
bromouracil, bromouridine,
5-hydroxydeoxyuridine,
and^-methyhnercapto-e-amino-S-hydroxymethylpyrimidine were essentially ineffective as inhibi
tors. The presence of 6-mercaptopurine
also pro
duced no marked inhibitions (Table 5).
5-Bromodeoxyuridine
and respiration and glycolysis.—Concentrations
of BrUDr which inhibited
the labeling of DNA-thymine
did not significantly
TABLE 4
affect cell respiration or glycolysis (Table 6).
S-BROMODEOXYUHIDINE
AND THE INCORPORATION OF
FORMALDEHYDE-C"
INTO ACID-SOLUBLE THYMINE
Incorporation of formaldehyde into protein and
COMPOUNDS
OFTUMORLLS147IN THEPRESENCE
OF free serine.—No inhibition by BrUDr on the in
THYMIDINE*
corporation of formaldehyde-C14
into protein or
T
TDr
TDrP
free serine was noted. Rather, there was a small
(total counts/mm)
increase in labeling (Table 7). These experiments
Control
1900
1320
430
further suggest that BrUDr does not intervene
BrTJDr
1940
1700
480
in cell metabolism
at the level of one-carbon
(0.99 millimoles)
* Each flask contained 5 Amólesthymidine, 2.5 /¿moles
glu- transfer reactions.
tamine, and 30 Amólesglucose. Total volume: 2.45 cc. There
was no deoxycytidine present.
Effect of 5-bromodeoxyuridine on the growth of
tumor E9514A.—Sixteen male C3H mice weigh-
TABLE 5
THYMIDINE
ANALOGS
ANDTHEINCORPORATION
OFFORMALDEHYDE-C"
INTOTHYMINE
COMPOUNDS
CONC.
(milli-
Tisera6C3HEDE9514ARat
DNA
ACID-SOLUBLE
TAG
(counts/mÃŒD//imole)
4930
2800
1970
7130
3480
2530
4330
3830
2370
5170
4140
3090
5920
4120
2680
TDr
TDrP
(total counts/min)
3710225023003290329517901700174014401
400047203670503040001190084807820130001120022400217002330028702210341026402350285
thymusRat
thymusADDITIONControlBrU*BrUBrUBrUControlHS-purine*MAHMP*ControlHS-purine*ControlMAHMP*MAHMPHOUDr*BrUR*mo!1
* Abbreviations: BrU = 5-bromouracil; HS-purine = 6-mercaptopurine; MAHMP = 2-methylmercapto-4amino-5-hydroxymethylpyrimidine; HOUDr = 5-hydroxydeoxyuridine; BrUR = 5-bromouridine; T = thymine;
TDr = thymidine; TDrP = thymidylate; A = adenine; G = guanine.
TABLE 6
5-BROMODEOXYUHIDINE
ANDRESPIRATION
OR
GLYCOLYSIS
OFLYMPHATIC
CELLS
ANDTUMORS
TABLE 7
5-BROMODEOXYURIDINE AND INCORPORATION OF
FORMALDEHYDE-C" INTO ACID-SOLUBLE SERINE
Qo,Control6.15.86.5BrUDr*6.05.76.8öSControl14.18.29.3BrUDr13.88.87.7ControlBrUDr*
ORCELLPROTEINBYTUMOR
6C3HED
TIBSÃœI
6C3HED
Spleen (mouse)
Thymus (rat)
* 3.91 Amólesbromodeoxyuridine added. All other flasks
contained 2.5 /¿molesglutamine, 1 /<mole deoxycytidine, and
80 //moles glucose. Total volume: 2.45 cc.
Counts/min/mg protein
Total counts/min serine
1370
60 ,800
1510
72,300
* 8.91 jumólesbromodeoxyuridine added. All flasks con
tained 1 yumoledeoxycytidine, 2.5 /jmoles glutamine, 30 /»moles
glucose.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
KIT et al.—5-Bromodeoxyuridine
ing 18-22 gm. were inoculated intraperitoneally with 1.5 X IO6E9514A tumor cells. Beginning
3 days after the injection of the tumor, ten of
the mice were given injections each day of phys
iological saline and six of the mice of BrUDr
(41.6 mg/kg body weight). Three of the control
mice died 14 days after receiving the tumor, three
at 15 days, one at 16 days, one at 17 days, one
at 19 days, and the last animal at 20 days after
receiving the tumor. Two of the treated mice
died at 16 days, one at 17 days, one at 18 days,
one at 19 days, and the final animal at 22 days
after receiving the tumor. The mean ( + the stand
ard error of the mean) survival of the control
and treated animals was 16 + 0.67 and 18 ±0.93
days, respectively. It may be seen that BrUDr,
when administered alone, produced a slight but
questionable prolongation of the survival time
of mice with lymphatic leukemia, E9514A.
DISCUSSION
The results reported in this paper suggest that
BrUDr represents a relatively specific antagonist
of the terminal steps of DNA-thymine synthesis.
The analog does not prevent the methylation
of deoxyuridine to form thymidine, the hydroxymethylation reaction leading to serine synthesis,
the anabolic reactions involved in the incorpora
tion of the isotopie precursor into protein, or
the energy-yielding processes of glycolysis and
respiration. The above interpretation is supported
by the preliminary findings of Eidinoff and coworkers (8). The latter investigators have reported
that BrUDr inhibited the incorporation of formate-C14, thymidine-C14, and orotic-C14 into the
DNA-thymine of human tumor slices. With the
latter substrate, DNA-cytosine and UNA uracil,
and cytosine were not significantly affected by
5 X 10~3M5-bromodeoxyuridine, despite the in
hibition of DNA-thymine labeling.
The interesting biological properties of BrUDr
and its demonstrable effects on the synthesis of
DNA-thymine in vivo or in vitro suggest that
further studies of this and related compounds
on the metabolism and growth of tumor cells
would be worth while. In the experiments de
scribed in the present paper, a mild biological
effect was observed during the growth of tumor
E9514A in C3H mice. An inhibitory effect on tu
mor growth in experimental animals by the related
compounds, 5-hydroxyuridine and 5-aminouridine,
has been reported (13), but at concentrations
of 3.6-10 times that used in the present experi
ments. 5-Bromouracil is an effective antibacterial
agent provided that sulfanilamide is added to
and Cell Metabolism
601
prevent de novo thymidine synthesis (1, 4, 11).
In neoplastic tissues, antifolic compounds can
be used to prevent the methylation of deoxy
uridine. It will be of interest to observe whether
the combined use of aminopterin and BrUDr
will prove more effective in prolonging the survival
of tumor-bearing animals than either alone (5).
SUMMARY
The effect of 5-bromodeoxyuridine and related
compounds on the metabolism of lymphatic leukemias LL5147 and E9514A, lymphosarcoma 6C3HED, rat thymus, and mouse spleen cells was
studied both in vivo and in vitro. It was observed
that 5-bromodeoxyuridine reduced the incorpo
ration of formaldehyde-C14 or formate-C14 into
DNA-thymine. The incorporation of the isotope
into DNA-adenine or guanine was affected only
at high concentrations of 5-bromodeoxyuridine.
Several compounds related to bromodeoxyuridine
had no significant effect. There was no inhibition
of the labeling of free thymine compounds, of
the hydroxymethylation reaction leading to serine
synthesis, of the incorporation of the isotopie
precursor into cell protein, or of cellular respiration
or glycolysis. A slight but statistically question
able increase of the survival time of mice bearing
ascites tumor E9514A was observed after the daily
administration of 41.6 mg/kg body weight of
5-bromodeoxyuridine.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
Effect of 5-Bromodeoxyuridine on Deoxyribonucleic
Acid-Thymine Synthesis and Cell Metabolism of Lymphatic
Tissues and Tumors
Saul Kit, Charles Beck, Odette L. Graham, et al.
Cancer Res 1958;18:598-602.
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