Download Binding of Daunomycin to DNA and the Inhibition of RNA and DNA

[CANCER RESEARCH 35, 1542-1546,June 1975]
Binding of Daunomycin to DNA and the Inhibition of RNA and
DNA Synthesis'
Nobuko S. Mizuno, Baiba Zakis, and Richard W. Decker
GeneralMedical Research,VeteransAdministration Hospital [N. S. M., R. W. D.], and Medical School, University ofMinnesota [N. S. M., B. Z.],
Minneapolis,
Minnesota 55417
SUMMARY
With synchronized tissue culture cells (L929), dauno
mycin had the greatest inhibitory effect on cell growth when
the drug was administered during the later stages of cell di
vision (late 5, G2, and M). The level ofbinding of daunomy
cm to DNA was not found to be influenced by the phase of
the cell cycle. The highest level of radioactivity from [3H]daunomycin was bound to DNA of the heterochromatin
fraction. Both RNA and DNA syntheses were inhibited in
isolated enzyme systems when daunomycin-treated DNA,
from which the unbound drug was removed by passage
through Sephadex column, was used. DNA polymerase was
reduced to one-fifth of the control activity, while that of
RNA polymerase was reduced to one-half. Similar experi
ments with daunomycin-treated
RNA
and DNA
polym
erase preparations showed that the drug had no effect on the
activities
of the enzymes
per se. Hence, the reduction
of
RNA and DNA polymerase activities could be accounted
for by the loss oftemplate activity ofthe drug-treated DNA.
Daunomycin caused a marked drop in the formation of a
complex between RNA polymerase and DNA, indicating
that the binding of daunomycin to DNA may give rise to
steric hindrance
effects that interfere with the association
of
the template to RNA polymerase enzyme. Sedimentation
profile in alkaline sucrose density gradient of DNA that had
been treated with daunomycin showed that no change in the
molecular weight could be demonstrated.
@
(NSC 82151) is an anthracycline
antibiotic
that has demonstrated antitumor activity against certain
animal and human neoplasms (22, 24). It was shown to in
hibit the synthesis of RNA and DNA in vivo (6, 14) and in
vitro (9, 23). With a low concentration of daunomycin, the
inhibition was irreversible with DNA polymerase but re
versible in the case of RNA polymerase (5). The drug
formed a stable complex with preformed DNA, presumably
by intercalating
between adjacent
base pairs of the double
helix (4, 13), and the biological activity of the drug is be
lieved to be related to this property. The purpose of this
study was to examine the factors influencing the binding of
1 Supported
in
part
by
USPHS
Grant
10298
from
the
National
Cancer
Institute. Part of the study was presentedat the 1974Annual Meeting of
the American Associationfor CancerResearchin Houston,Texas.
ReceivedNovember21, 1974;acceptedMarch 11, 1975.
1542
MATERIALS
Materials.
AND METhODS
[methyl- ‘4C]Thymidine (specific
activity,
54.2 mCi/mmole),
[3H]TTP (specific activity, 28.4 Ci/
mole), and [3HJCTP (specific activity, 28.4 Ci/mmole)
were from New England Nuclear, Boston, Mass. [methyl
sHjThymidmne (specific activity, 14.1 Ci/mmole) was from
ICN, Cleveland, Ohio. [3H]Daunomycin (specific activity,
69.5 mCi/mmole) was a gift from Farmitalia, Milan, Italy.
RNA polymerase from Escherichia coli B (EC 2.7.7.6; 294
units/mg protein) and DNA polymerase from Micrococcus
lysodeikticus (EC 2.7.7.7; 90 units/mg protein) were from
Miles Laboratories,
Elkhart, md. Calf thymus DNA was
from Worthington Biochemical Corp., Freehold, N. J.
[3H]DNA was prepared from L-cells that had been incu
bated for 24 hr with [3H]thymidine (final concentration, 0.5
@zCi/ml). The procedure of Kirby and Cook (15) was used
for isolationof DNA. The specific activity of [3H]DNA was
2170 cpm/@g. Daunomycin-treated
[3HJDNA was prepared
by reacting [3HJDNA (27.5 zg) with daunomycin ( 1.4 zg)
in 1.0 ml of H 20 for I hr. then the unbound drug was re
moved from the DNA by repeated dialysis in Minicon A-25
Concentrator (Aminco Corp., Lexington, Mass.). The spe
cific activity of DNA was 2180 cpm/@ig and was unaltered
by this treatment.
Methods. Mouse fibroblast tissue culture cells (L929)
were maintained in suspension culture in Joklik-modified
INTRODUCTION
Daunomycin
daunomycin to DNA and to investigate the mechanism
whereby the drug exerts its inhibitory action on the synthe
sis of RNA and DNA.
Eagle's minimal essential medium (Grand Island Biological
Co., Grand Island, N. Y.) supplemented with 10% fetal calf
serum and 2 mM glutamine. For cell synchrony studies, the
cells were reversibly arrested in G by maintaining them in
isoleucine-deficient medium for 96 hr (20). When isoleucine
was restored to the medium, DNA synthesis was initiated
with a high degree of synchrony, reaching a maximum rate
at 12 to 16 hr. The generation time was 20 to 24 hr. In order
to determine whether the cytotoxic effects of daunomycin
varied during the cell cycle, the cells from different stages of
the cell cycle were incubated with daunomycin (2 x l0' M)
for 1 hr. The cells were then washed and transferred to fresh
medium. After incubation for 24 hr. cell counts were made
for assessment of cytotoxicity.
In order to investigate whether the extent of binding of
daunomycin
to DNA was influenced
CANCER
by the cell division
RESEARCH
VOL.35
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Daunomycin
Binding to DNA
cycle, synchronized cells at various stages of the cycle were and counting in the liquid scintillation counter.
To determine whether daunomycin had any effect on ei
reacted for 30 mm with [3Hjdaunomycin (1.6 x l0@ .M).
[‘4C]Thymidinewas added to a final concentration of 10 ther RNA or DNA polymerase enzymes, purified enzyme
zCi/ml to simultaneously measure DNA synthesis. The preparations (60 to 75 @g)were reacted with daunomycin
cells were then washed with Hank's balanced salt solution
(Grand Island Biological Co.). DNA was precipitated with
0.2 N perchloric acid, and the resultant precipitate was
washed twice with the same acid. RNA was hydrolyzed by
heating the precipitate in 0.3 N KOH at 37°for 1 hr. DNA
was isolated by adding perchloric acid and centrifuging.
(4 ;@gin 0.4 ml of 0.01 Tris, pH 8.0) for I hr at room tern
perature.
Then the unbound
drug was removed
dialysis in Minicon A-25 Microconcentrator.
by repeated
Enzyme con
centrations were adjusted to the original values by absorb
ance measurements at 280 nm. The enzymes were assayed
as described above. Control enzymes were treated in the
DNA was extracted from the precipitate by heating it in 10% same manner for the drug treatment.
The method of Jones and Berg (I 1) was used to deter
NaCI (pH 7.5) for 30 mm at 100°.DNA was quantitated by
mine whether daunomycin treatment of DNA affected its
absorbance measurements at 260 nm, and the radioactivity
binding to RNA polymerase.
In this procedure,
RNA
was measured by adding 1 ml of extract to 10 ml of Triton
X-lOO:PPO:POPOP:toluene
counting
(450 ml:4 g:0.8 g:l000 ml) and
in a liquid scintillation
counter
polymerase and DNA (either untreated or treated with
(1 1). Dual-label
daunomycin) were separately filtrable through Millipore
technique was used for estimation of 3H and ‘4C
counts.
membrane filter, but the combination of the 2 was quantita
tively retained by the filter. Incubation mixture (0.25 ml)
Binding of daunomycin to DNA of various cell fractions
contained I @tmoleMgCI3, 3 @tmoles2-mercaptoethanol,
10
was studied in cells that had been incubated with [3H]dauno
@imoles Tris-CI, pH 8.0, 2 zg untreated or daunomycin
mycin (1.6 x l0@ M) for I hr at 37°.The cells were washed
in 0.14 M NaC1:5 m@iKCI:7 m@iNa2HPO4:25 m@iIris, pH treated [3H]DNA (14,500 cpm), and 0.5 to 8.5 sg of RNA
7.5, and suspended in 10 volumes of 10 mr@iTris:l m@i polymerase. After standing for 5 mm at room temperature,
EDTA:l0 mM NaCI, pH 7.5. After 10 mm, the cells were the mixture was filtered through Millipore (Type HA, Milli
homogenized with 25 strokes of the Dounce homogenizer. pore Corp., Bedford, Mass.). The filters were washed with
To the homogenate from 2 x l0@cells, 1.0 ml of 1.5 M 40 ml of0.05 M NaCl:0.0l M Tris, pH 8.0, and after drying
sucrose was added, and the mixture was layered over 6.0 ml they were placed in vials containing 10 ml PPO:POPOP:tol
of 1.5 N sucrose in 10 m@i Tris: I m@i EDTA:lO mr@i uene (4 g:0.3 g: 1000 ml) and counted in a liquid scintillation
NaCl, pH 7.5, and centrifuged at 30,000 rpm in Spinco counter. For negative controls, assays were made with an
enzyme preparation that had been denatured with heat (70°
SW 41 rotor. The crude mitochondrial fraction that sepa
rated at the interface was purified according to Kasamatsu for 5 mm).
For analyses by alkaline sucrose density gradient centrif
et a!. (12). The nuclei fraction that sedimented to the bot
torn of the tube was sonically extracted for 70 sec (Branson ugation, DNA was isolated as previously described from
Sonifier, 5-75, setting of 2), and euchromatin,
hetero
tissue culture cells that had been maintained for I hr with
chromatin, and an intermediate chromatin fraction were daunomycin (1.8 x l0@ M). DNA (400 .tg in 0.2 ml of 5%
isolated according to the method of Yasmineh and Yunis sucrose) was layered on top of4.5 ml of sucrose gradient (5
(25). DNA was isolated from each cell fraction, and spe
to 20%, w/v, at pH 12.7). The gradients were centrifuged at
cific activity measurements were made as before.
34,000 rpm in Spinco No. 40 rotor for 2.5 hr at 20°.The
To study the template activity of the drug-treated DNA, bottoms of the tubes were punctured and 3-drop fractions
were collected, to which 0.5 ml of H2O was added. The ab
calf thymus DNA (0.5 mg) was reacted with daunomycin
sorbance was read at 260 nm.
(25 jsg) in 1.0 ml of I mM MgC12 for 16 hr at room tempera
ture. Uncombined drug was removed by chromatography
on a column (I .4 x 25 cm) of Sephadex G-50, using 0.01 M
potassium phosphate, pH 7.0, as eluant. Fractions contain
ing DNA were pooled, lyophilized, and dissolved in a suita
@
ble volume for template assay. Alternatively, unbound drug
was removed by repeated ultrafiltration in Minicon A-25
Microconcentrator.
Control DNA was treated in the
manner except for the omission ofthe drug treatment.
polymerase was assayed according to Lynch et a!.
using a purified preparation from M. lysodeikticus.
polymerase was determined according to Burgess (3),
same
DNA
(I 6),
RNA
using
the RNA polymerase preparation from E. coli B. At the end
of the incubation
periods, aliquots of the reaction mixtures
were transferred to glass fiber discs (Whatman GF/C) and
the nucleic acids were precipitated with perchloric acid.
The discs were washed and dried and allowed to stand over
night with 0.25 ml of NCS Solubilizer
(Amersham/Searle
Corp., Arlington Heights, Ill.). Radioactivity was deter
mined by addition
of 10 ml of liquid scintillation
mixture
RESULTS
AND DISCUSSION
DNA synthesis and cell division were synchronized in L
cells by restoring isoleucine to cells that had been brought to
a state of
arrest by growth in an isoleucine-deficient me
dium. The influence ofcell cycle on the cytotoxicity of dau
nomycin was studied by measuring growth of cells that had
been exposed to a fixed dose of the drug at various time in
tervals after isoleucine refeeding. The results showed that
daunomycin caused a depression of cell counts throughout
the cell cycle and that the maximum effect on cell growth
occurred when the drug was added during the later stages of
cell division (late S. G2, and M). These findings were con
sistent with those obtained with other mammalian cell sys
terns, in which the maximum effect ofthe drug was observed
in late S period (14, 18). In Chinese hamster cells, dauno
mycin at low doses showed little effect on the entry of cells
into S but was effective in preventing cells from reaching
JUNE 1975
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1543
N. S. Mizuno
et al.
Table I
Binding of[3H]daunomvcin
to DNA ofcell fractions
mitosis (19). A delay in G2 period occurred when daunomy
cm was added to cultured human leukocytes (1). It is not
clear whether the antimitotic activity of daunomycin is de
DNAMitochondria15.9Euchromatin9.9Heterochromatin36.8Intermediate
Cell fractionpmoles
daunomycin/mg
rived exclusively from the known property ofthis compound
to bind to DNA and thus interfere with the biosynthesis of
various macromolecules.
Cells at various stages of the cell cycle were reacted with
a fixed dose of [3H}daunomycin, and then DNA was ex
tracted for study. The radioactivity bound to DNA (ex
pressed as pmoles daunomycin per mg DNA) showed little
variation throughout the cell cycle (Chart 1). Using auto
radiographic technique, Silvestrini et al. (17) showed that
maximum uptake of [3Hldaunomycin
by rat fibroblasts
I .I
chromatinI
Table 2
Effect ofdaunomvcin
on RNA and DNA polvmerase activities
Activity: %of
control'DNARNADN
took place at late S stage; however, our findings did not in
dicate that the level of binding of the drug to DNA was in
APolymerasepolymerasepolymeraseDaunomycin
fluenced by the phase ofthe cell cycle.
Untreated25.3
1.9treatedUntreatedDaunomycin
Cells that had been treated with [3H]daunornycin were
homogenized, and DNA was extracted from the following
structures:
mitochondria,
euchromatin,
heterochromatin,
and an intermediate chromatin fraction. From specific ac
tivity measurements of DNA, it was shown that the highest
level of binding of 3H (estimated as pmoles daunomycin per
mg DNA) was found to be associated with the heterochro
matin fraction (Table I). Lesser amounts were found to be
present in DNA from the other structures. The significance
of preferential
binding of daunomycin
to DNA of hetero
chromatin fraction remains obscure at present. Ward et a!.
(23) reported
that daunomycin
inhibited
DNA-dependent
RNA synthesis regardless of the base composition of the
DNA templates used; however, recently, Zunino et a!. (27)
have shown that adenine-thymine-containing
synthetic
DNA templates were more sensitive to the action of dauno
mycin. Mouse heterochromatin has been shown to be
enriched with adenine-thymine-rich
satellite DNA (26).
Sensitivity of cells to the toxic effects of daunomycin at the
C.,
a
-@
E
C
.4
±I
±15.8109
±13.0
treated112
a Values
obtained
with
untreated
DNA
and
untreated
polymerase
under
identical conditions ofassay were taken as controls (100% activity). Results
are means of 2 experiments ± @.
E.
end of S may be related to the fact that DNA-containing
poly(dA dT) sequences are being replicated at this time (8).
Binding of daunomycin to DNA is believed to be related
to the inhibitory effect of the drug on RNA and DNA syn
thesis. Daunomycin derivatives with reduced binding capac
ity, as measured by their effects on sedimentation velocity
and relative viscosity of DNA, were found to have corre
spondingly less effect on the inhibition of nucleic acid syn
theses and on mitotic index (7). At low concentrations
of
daunomycin, DNA synthesis was more inhibited than was
RNA synthesis (9, 14). However, it has been shown that the
inhibition
of DNA synthesis was irreversible,
while that of
RNA was initially depressed but gradually returned to con
trol levels (2, 5). Therefore, the relative activity of dauno
mycin on incorporation of precursors into RNA and DNA
in vivo may depend upon the time of observation after the
drug administration.
Inhibition of RNA and DNA synthesis has been observed
when daunomycin was added to isolated enzyme systems (9,
10, 23, 27). In order to distinguish between the effect of the
drug on the primer and on the enzyme, daunomycin-DNA
0
0
C
complex was prepared, from which unbound daunomycin
was removed by passage through a Sephadex column. With
C
.°
@0
C
0
a
0
E
0
C
0
Z4
I
hoursaffirisotaucine
r.faading
Chart 1. Uptake of ‘Hby DNA when cells at various intervals after
isoleucine refeeding are treated for 30 mm with [‘H]daunomycin (1.6 x
l0' M). In order to monitor DNA synthesis, [“C]thymidinewas added to
a final concentration of 10 pCi/ml. DNA was isolated and analyzed as
described in the text. Data were estimated by assuming parity between spe
cific activity ofthe drug and that ofthe moiety that boundto DNA. Points,
meanfrom 2 experiments.Bars,SE.
1544
± 3.754.0
daunomycin-treated
DNA as primer, the activity was re
duced to 25% of control value in DNA polymerase system
and to 54% of control in the case of the RNA polymerase
system (Table 2). On the other hand, when enzymes that
were similarly treated with daunomycin were used in the
assays, no reduction of polymerase activity was observed.
Hence, daunomycin treatment of DNA caused a loss of
template activity presumably by its binding to DNA. A sim
ilar conclusion was reached by Zunino et a!. (27), who were
to overcome the inhibition of RNA polymerase by dauno
mycin by an increase in DNA concentration. These studies
indicated that reduced template activity of DNA could be
responsible for the inhibition of RNA and DNA polymer
ase activity by daunomycin.
CANCER RESEARCH VOL.35
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Daunomycin
2000
.
i__c
I
E
1000
I
I
I
_
Daunomycin reduced the sedimentation coefficient of
DNA in CsCI gradient (4, 13). This shift in buoyant density
suggested a modification of molecular size or shape of DNA
—0 ——————
I
in solution. The sedimentation
@
profile of DNA from dauno
mycin-treated cells in alkaline sucrose gradient (5 to 20%,
w/v, pH 12.7) was shown to be unchanged from that of con
I
U
Binding to DNA
-
- -
trol cells (Chart 3); thus, no gross modification
of molecular
weight could be demonstrated by this technique.
ACKNOWLEDGMENTS
,449RNA poIymeras@
@
The authors are grateful to Farmitalia,
Milan, Italy, for a generous gift
Chart 2. Effect of daunomycintreatment of [‘H]DNAon its ability to of tritiated daunomycin.
associatewith RNA polymerase. Mean
SE. between2 experiments
averaged±6.9%.•,untreated [‘H]DNA+ denaturedDNA polymerase;
0, untreated [‘H]DNA+ undenaturedRNA polymerase; @,
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Chart 3. Alkaline sucrosedensity gradient sedimentation profiles of
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CANCER RESEARCH VOL.35
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Binding of Daunomycin to DNA and the Inhibition of RNA and
DNA Synthesis
Nobuko S. Mizuno, Baiba Zakis and Richard W. Decker
Cancer Res 1975;35:1542-1546.
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