[CANCERRESEARCH39, 1321-1327,April 1979]
0008-5472/79/0039-0000$02.O0
Differential Effect of Adriamycin on DNA Replicative and Repair
Synthesis in Cultured Neonatal Rat Cardiac Cells1
Henie Fialkoff, Myron F. Goodman,2 and Maria W. Seraydarian
Department of Biological Sciences, Molecular Biology Section, University of Southern California, University Park, Los Angeles, California 90007(H. F.,
M. F. G.J, and Center for Health Sciences, School of Nursing, Universityof California, Los Angeles, Los Angeles, California 90024 (M. W. S.!
ABSTRACT
The effect of the potent antitumor antiobiotic Adriamycin
(ADM) on DNA replication
and unscheduled
DNA synthesis
in cultured rat cardiac cells was investigated. Autoradiog
raphy and [3H]thymidine incorporation studies were carried
out on parallel cultures. DNA replication was depressed for
up to 6 days following a 3-hr pulse of ADM administration.
An ADM concentration of 1 .tg/mI which was effective in
reducing replicative DNA synthesis by as much as 75% did
not reduce the ability of cardiac cells to repair UV-damaged
DNA. However, cells exposed to higher ADM concentrations
failed to undergo significant UV-induced repair. In the
absence of UV treatment, ADM did not stimulate unsched
uled DNA synthesis. To accountforthe differential response
of the cardiac cell cultures to replicate and repair DNA, we
propose that ADM exerts a localized effect on DNA synthe
sis covering a region proximal to its primary intercalation
site.
INTRODUCTION
The anthracycline antibiotic ADM3 has been shown to be
effective as a chemotherapeutic agent in the treatment of a
variety of human tumors (2). Its success in retarding the
growth of rapidly dividing malignant cells has been largely
attributed to the ability of anthracycline drugs to intercalate
between the parallel stacked bases of DNA (1, 8, 29). The
resultant malformation in the double helix may be respon
sible for the inhibition of subsequent DNA and RNA synthe
sis (15, 21) and is implicated in the effectiveness of ADM in
arresting tumor growth.
In spite of the antitumor value of ADM, its clinical admin
istration has been restricted because of an associated
cardiotoxicity (25, 27). Investigations on cardiotoxicity in
duced by ADM and related drugs have shown that gross
morphological, physiological, and molecular changes oc
cur in rat tissue culture (32) as well as in rats (9, 23) and
other animal models (16-18, 20).
In the current study, we report that (a) neonatal mat
myocardial cells in culture are capable of unscheduled DNA
synthesis in response to UV irradiation, (b) ADM, at a
concentration of 1 @g/ml,does not appear to affect the rate
or extent of unscheduled DNA synthesis under conditions
I
Supported
by
NIH
Grants
CA17358
and
GM21422
and
grants
from
the
University of California Cancer Research Coordinating Committee and Amer
can Heart Association, Greater Los Angeles affiliate.
2 To
3 The
whom
requests
abbreviations
for
used
reprints
are:
should
ADM,
be
addressed.
Adriamycin;
F-1O
medium,
Ham's
F-
10 medium plus 15 mM NaHCO,- 1 mM CaCl,-penicillin (10@ units/
liter)streptomycin (0.1 g/liter); HU, hydroxyurea; dThd, thymidine; HC, heart
control.
Received May 15, 1978; accepted January 12, 1979.
where meplicative DNA synthesis is inhibited significantly,
(c) repair synthesis occurs in virtually the entire cell popu
lation; and (d) at concentrations greater than 2 @tg/ml,
ADM-treated cells have lost the ability to repair DNA in
response to UV-induced DNA damage.
MATERIALS
AND
METHODS
Neonatal (1 to 4 days old) Sprague-Dawley rats were
supplied by Simonson Laboratories (Gilroy, Calif.); F-iO
medium and fetal calf and horse sera were purchased from
North American Biologicals, Inc. (Miami, Fla.); HU was
purchased from Sigma Chemical Co. (St. Louis, Mo.);
[3H]dThd was purchased from Schwarz/Mann (Orangeburg,
N. Y.), had a specific
activity of 62 Ci/mmol,
and was used
at a concentration of 10 pCi/mI unless otherwise noted.
ADM, a gift of Dr. N. R. Bachur of NIH, was first dissolved in
sterile distilled H2Oand then diluted to the required concen
tration in F-iO medium. All photographic supplies such as
the NTB autoradiographic emulsion, D-19 developer, fixer,
and Ektachrome 160 film were purchased from Eastman
Kodak Co. (Rochester,
N.Y.).
Preparationof CardiacCultures.Heterogeneouscardiac
cultures were prepared by a modified method of Harary and
Fanley (14), plated in F-b medium supplemented with 10%
fetalcalfand 10% horse sera,and grown in a water
saturated 37°,95% air-5% CO2 incubator. Such cultures
contained spontaneously contracting cardiac m uscle cells
and quiescent epithelioid or nonmuscle cells. The 2 cell
populations could be separated into greater than 80% pure
myocardial cultures and greater than 95% nonmyocardial
cultures. Advantage is taken of the faster mateof attachment
of nonmyocardial cells to the dish. Following one hr of
incubation, the medium containing the unattached myocar
dial cells is transferred to a new culture dish. The transfer
procedure is then repeated after an additional 1 hr incuba
tion. The cells attached to the first dish comprise the
cardiac-derived, nonmuscle cell culture. The particular cell
population type is characterized on the basis of cell density
and beating and staining characteristics (32), thus indicat
ing the physiological as well as the morphological integrity
of the cells. During this study both homogeneous' ‘
and
heterogeneous
cultures
were
investigated.
However,
since
preliminary experiments showed no significant difference
in the synthesis
ability
of the various
cell types,
the respec
tive results have been pooled unless otherwise noted.
ADM
AdministratIon.
At 18 hr in culture,
one-half
of the
cellculturewas
placed in 4 mlofi,
2, 5, ori0 @.tg
of ADM
per ml of F-i0 medium for 3 hr. After this exposure, HG and
ADM-treated plates were rinsed once in F-iO medium,
returned to 4 ml of supplemented F-iO medium, and rein
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1321
. H.
Fialkoff
et
a!.
cubated for the desired periods.
POPOP in 50 ml dioxane
(11). Again, the slides were dried
DNA ReplicationAssay. At specificintervalspost-ADM in the dark, sealed with a dessicant in a light tight box, and
treatment, both HG and ADM plates were exposed in dupli
cate to [3HJdThd for 0, 90, and 180 mm. At the end of the
exposure time, the plates were rinsed 5 times in 0.9% NaCI
solution, and 2 ml of 0.3 M perchlonic acid were added to
the plates.The cellular
materialwas scraped from the
plates and centrifuged at 14,000 rpm in a Sorvall type SS 34
rotor at 4°for 20 mm. The supemnatant was discarded, and
the pellet was suspended in 0.5 ml of 0.5 M perchlonic acid,
heated for 15 mm at 70°to extract the DNA, and centrifuged
for 30 mm. The supemnatant was saved, and the DNA
extraction was repeated. The supemnatants were pooled
and brought up to a final volume of 1.0 ml. A 0.5-mI sample
was counted in 9 ml of a scintillation mixture containing 2
i@oftoluene,1 e ofTritonX-100,16.5g of PPO and 0.375g
of POPOP. Protein was determined on the pellet using the
method of Lowry et al. (26).
DNA
Repair
Assay.
At 68 hr in culture,
HG and
ADM
plates were UV irradiated for 0 to 100 sec. It had been
previously determined that at a distance of 4.5 cm above
the exposed cells, a UVS-54 germicidal lamp delivered an
incident dose of 15 ergs/sq mm/sec at 254 nm. Three ml of
stored at —70°
for 2 weeks. The sealed slides were brought
to room temperature in a vacuum desiccator, developed
with Kodak D-19 (diluted 1:2 with deionized H20), and fixed
with Kodak fixer. After a deionized H2O rinse, the slides
were stainedfor7 mm ina bufferedGiemsa stain(10ml
Giemsa stock, 6 ml 0.1 M citric acid, 6 ml 0.2 M disodium
phosphate, 6 ml methanol, diluted to 200 ml with deionized
H20) and then dipped in a citrate buffer (1 ml 0.1 M citric
acid, 1 ml 0.2 M disodium phosphate, and 48 ml deionized
H,O). The slides
were
mounted
with
Permount
(Fisher
Scientific) and photographed under a Leitz Ortholux
microscope using Ektachrome 160 film.
II
RESULTS
To elucidate the effect of ADM on the synthesis of DNA by
cardiac cells in culture, the incorporation of [3HJdThd into
DNA both in the presence and absence of ADM was deter
mined. Chart 1 shows that at every age tested, there is an
essentially linear incorporation of [3H]dThd during the 3-hr
exposure period both in the presence and absence of ADM.
F-10 medium containing [3H]dThd (10 p@g/ml)either with or However, at each time point, dThd incorporation normal
without 0.1 M HU were added to each plate immediately
ized to protein concentration is reduced significantly in
after UV exposure. A 4-hr incubation was followed by five ADM-treated cultures. Attempts to measure DNA content by
0.9% NaCI solution rinses and the precipitation of proteins
the assay of Burton (4) were unsuccessful because an ADM
and nucleic acids by 2 ml of cold 10% trichloroacetic acid. treated culture plate contained a reduced number of cells
The cellular material was scraped off and collected on and, therefore, insufficient DNA for the assay. Thus, protein
Whatman GF/A glass filter discs mounted on a vacuum determinations were chosen as a reproducible parameter
suction flask. The discs were dried for 20 mm under an IR against which to standardize [3H]dThd incorporation into
lamp and counted in 8 ml of toluene fluor, a scintillation
DNA. The possibility existed that a cell could increase in
mixture containing 3.79 f toluene, 379 mg POPOP, and size without a concomitant increase in DNA. However, there
18.95 g PPO. Protein determinations were performed on proved to be a direct correlation between the average
parallel plates from the same culture. Irradiation did not number of cells per field and the protein content per plate
(data not shown). It is apparent from the cell number per
measurablyalter
theproteinlevels.
Autoradiography.Trypsinizedcardiaccellswere seeded plate that ADM has effectively caused a cessation of cell
onto a glass slide placed in a 100 x 20-mm tissue culture division, probably due to the inhibition of DNA synthesis. At
dish. The media and growth conditions of the culture were all points studied, incorporation of [3HJdThd per j.@gprotein
as previously described. However, because cardiac cells do was greater in the HG cells than in the ADM-tneated ones.
not attach as readily to glass as to plastic, an additional 48 The latter reached and maintained a nearly steady low level
of incorporation within a day after treatment. As the cul
hr were allotted prior to ADM treatment. Thus the slide
grown cardiac cultures were 66 hr old when exposed to a 3- tures aged, the dramatic differences between the 2 syn
hr pulseof ADM (0,1,2, 5, or 10 @g/ml).
At 114 hr,the thetic abilities lessened, possibly because the control cul
platesused forrepairsynthesis
were treatedwithUV irma tures were now confluent and replicated infrequently. The
diation (675 ergs/sq mm) as described above for the DNA ADM cells never reached confluency within the time course
repair assay. Three ml of F-10 medium containing 0.1 M HU of this study. Therefore, only ADM and not contact inhibi
and 10 @Ci
[3H]dThd per ml of F-10 medium were added to tion was responsible for decreased synthesis.
Thymidine incorporation in Chart 1 is indicative of total
the irradiated plates immediately following UV exposure.
Control samples used for replication were unirradiated and DNA synthesis and does not distinguish between replicative
treated with 3 ml F-10 medium containing 10 @Ci
[3HJdThd and repair synthesis. To investigate the effect of ADM on
per ml. Following a 4-hr incubation, the slides were fixed in DNA repair required the visualization of repair synthesis
a freshly prepared methanol:acetic acid (3:1) mixture for and the concurrent inhibition of replication. Increasing the
three 5-mm periods and then rinsed in 70% ethanol for an dose of UV irradiation depresses replicative synthesis, but
additional three 5-mm periods and aim-dried. Kodak NTB the level of DNA repair is so low as to still be masked.
emulsion, which had been previously melted for 1 hr in a Therefore, irradiating the cells with UV in the presence of
450
water
bath,
was
thoroughly
mixed
in
the
dark
with
an
equal amount of deionized H2O. The fixed slides were
dipped in the emulsion and dried for 1 hr in the dark. Then
the slides were dipped for 10 sec in a freshly prepared
scintillation mixture composed of 0.35 g PPO and 10 mg
1322
HU permits DNA repair to be more readily observed.
HU is
known to interfere with the reduction of ribonucleotides in
the formation of DNA precursors (35), thus nearly eradicat
ing replication while allowing repair synthesis to continue
(10). A 0.1 M concentration
of HU reduced
replicative
CANCERRESEARCHVOL. 39
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Effect of ADM on DNA Replication and Repair
z
(U
I0
a.
Chart 1. Incorporation of [3H]dThd (specific activity,
62Ci/mmol) into DNAof culturedheterogeneouscardiac
cells from neonatal rats as determined at (a) 21, (b) 40,
(c), 88, and (d) 178 hr of culture growth. Points, incor
poration of PH]dThd per @&g
protein. Samples were
assayedas describedin “Materials
and Methods―
at 0,
90, and 180 mm of [3H]dThd incorporation at all ages
indicated. ADM was administered as a pulse (1 @.tg/ml)
at
18to21 hr. 0,HC;•,ADM.
a.
C.)
z
0
I4
0
a.
0
C.)
z
.5
.5
0
90
80
0
90
180
[ @HJdThd
PULSE
LENGTH
(mln)
synthesis by 95% (see Chart 2, inset) yet allowed UV-stimu
lated synthesis to be observed over background replication
levels which were designated as 100% activity (Chart 2).
Although there was a great deal of experimental variation in
the respective repair abilities of HC- and ADM-treated cul
tures, it is obvious that at all the UV doses investigated,
stimulation over background levels was present. Enhanced
repair synthesis is evident as increased [3H]dThd incorpo
ration due to the excision and subsequent repair of the
pyrimidine dimers produced by UV irradiation. Such synthe
sis occurred up to exposures of 1500 ergs/sq mm. However,
since this dose resulted in some morphological changes,
675 ergs/sq
mm served as a maximum
dose. The possibility
of photoreactivation (33) in rat cardiac cells was unlikely
since identical experiments run in the presence and ab
sence of visible light failed to indicate any diftemence in
[3H]dThd incorporation
(data not shown).
The effect of UV exposure on DNA replication and repair
in ADM-treated and untreated cells is presented in Table 1.
At a concentration
of 1 j@g/ml, ADM caused a 75% reduction
in replicative synthesis, yet had essentially no effect on the
stimulation of UV-induced repair synthesis. At higher con
centrations of ADM (2 and 5 @g/ml),a lower level of induced
repair synthesis may possibly be elicited, while at the 10
@ig/mllevel of ADM, HU-affected [3H]dThd incorporation
completely failed to respond to UV stimulation.
Initially, it was assumed that since epithelial cells divide
more frequently than do myocardial cells, epithelial cells
would rapidly comprise the major subpopulation of a het
erogeneous cardiac culture and obscure any DNA synthesis
activity undertaken by myocardial cells. Indeed, in terms of
[3H]dThd incorporation
per
@g
protein,
both in the presence
and absence of ADM, epithelial cells carried out replication
better than did myocardial cells (data not shown). However,
with respect to percentage of changes in activity, myocar
dial cells frequently did as well as or better than their
epithelial counterparts. For example, the detrimental effect
of ADM on [3H]dThd incorporation, the decrease in replica
tion by UV exposure, and the ability to repair damaged DNA
are similar in the 2 subpopulations. With regard to total
[3H]dThd incorporation and percentage of activity, hetero
geneous cultures generally reflected epithelial cell condi
tions, but this was not always the case. The great deal of
overlap among synthesis abilities of the 3 culture types did
not warrant separate figures for [3H)dThd incorporation
data. Therefore, all the results have been pooled for each
parameter investigated (Charts 1, and 2; Table 1).
The above results did not eliminate the possibility that the
low [3H]dThd incorporation seen in HU-treated cultures may
actually be due to a small amount of residual DNA replica
tion and not to repair synthesis. To prove that substantial
repair synthesis is occurring in the presence of HU, we
studied the incorporation of [3H]dThd in individual cells by
means of autoradiography. Dark grains in the cell nucleus,
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1323
H. Fialkoff et al.
of unimmadiated
ADM-tmeatedcells(Fig.1,C, E, G, and I)
show no indication of excision-repair induction. Thus, at
the levels of ADM tested, there was no evidence of concom
itant excision repair associated with possible ADM-induced
strandbreakage.
Since the inhibition of DNA synthesis by ADM is believed
to occur during S phase (19, 22), the possibility existed that
only those cells which were in the S phase would die.
Unaffected cells which escaped the action of the drug could
then proceed to synthesize DNA normally. This is not likely
since we are able to observe fluorescence specific for ADM
l80-@
I60—
z0
@
l40@0
1
@I20ç@@
(12) in all myocardial
I-. IOOi
U
0
80@
z
0
I-
4
60@
I(a
40@—
-J
@
I
20
@
ri
0
I
50
I
450
300
I
600
I
750
Chart2. Effect of UV irradiation on DNAreplication and repair in ADM
treated and untreated cardiac cultures. Incorporation of [3H]dThd (specific
activity, 62 Ci/mmol) was measured following a 4-hr pulse in the presence or
absence of 0.1 M HU. Bars, 1 S. E. [ADM], 1 pg/mI pulse at 18 to 21 hr. The
data are represented as the stimulation of [3H]dThd incorporation by UV
irradiation compared to an unirradiated base line arbitrarily indicated as
0,
HC; •ADM;
&
HC plus
HU; A, ADM
plus
HU. Inset,
effect
of HU
on replicative synthesis. At 68 hr of culture age, HC samples were exposed
to [‘H]dThd
for 4 hr in the presence of 0 to 2 M HU. Radioactivity and protein
were measured on parallel samples from the same culture. Points, individual
samples.
indicating the incorporation of [3H]dThd, appear uniformly
in 100% of the cells treated with UV and HU both in the
presence and absence of ADM treatment (Fig. 1, B and D).
In contrast, under meplicative conditions, cultures had nu
merous grains in approximately 10% of the HG cells and 2
to 5% of the ADM-treated cells. The vast majority of cells
contained no detectable grains (Fig. 1, A and C).
Although some minimal [3H]dThd incorporation does oc
cur in the presence
of HU in 2, 5 and 10
@g/ml-treated
cells
(Table 1), this appears to be a background level of repair
and is not stimulated by UV irradiation. The possibility of
such a base line level is further supported by autoradi
ographic studies which indicate that under repair assay
conditions several grains are present in all the nuclei of
even ADM (10 @tg/ml)-treatedsamples (Fig. 1J). In summary,
it seems as if UV-stimulated
repair
synthesis
is severely
limited
atADM concentrationsgreatenthan
1 @g/ml.
Since ADM has been observed to cause DNA strand
breakage (6, 31) and various chromosomal abnormalities
(34), it was conceivable that ADM could stimulate repair
synthesis itself. Since it is possible that any repair of ADM
damaged DNA may have occurred in the interim between
drug exposure and [3H]dThd incorporation analysis, we
looked at repair synthesis immediately following a 3-hr ADM
treatment in the absence of UV irradiation. HG cells incor
porated [3H]dThd at a level of 23.6 ±4.6 cpm/@g (S.E.) of
protein while cells treated with ADM (1 j.@g/ml)incorporated
26.3 ±9.6 cpm/@g of protein.
1324
cells irrespective
DISCUSSION
uv IRRADIATION
INTENSITY
(ergs/sq
mm)
100%.
and nonmyocardial
of cell phase almost immediately after ADM exposure.
Although neither the activity nor the intracellular concentra
tion of ADM was determined, a drug-induced fluorescence
related to either ADM or possibly one of its metabolites is
present in the majority of the cardiac cells up to at least 48hr post-ADM administration (data not shown). Thus, we
conclude that UV-induced repair synthesis taking place in
the presence of ADM cannot be attributed solely to cells
which have managed to completely escape drug action.
In addition,
autoradiographs
The use of ADM as an antineoplastic agent has been
limited by an associated cumulative, dose-dependent car
diomyopathy. Since ADM's effectiveness is linked to its
ability
to destroyselectively
the proliferating
cellscharac
teristic of tumor growth, it is not clear why cardiac cells,
which replicate at a very slow rate if at all, are also
damaged. In vivo studies using ADM have suggested soy
eral possibilities for its deleterious action in nondividing
cells. For example, the drug may cause the formation of
free radicals with resultant lipid pemoxidation and cellular
damage (28). Since it is well documented that ADM can
both intercalate into DNA (29) and cause single-strand
breaks
(6, 31 ), numerous
studies
have been concerned
with
impairment by ADM of nucleic acid synthesis (15). We
pursued this line of reasoning by investigating the effect of
ADM on the 2 facets
of DNA synthesis,
replication
and
repair.
In the former case (Chart 1), ADM (1 @g/ml)reduces DNA
replication in cardiac cells at every age tested with the
reduction being most significant during the rapid growth
phase (—‘30
to 120 hr). Yet, in spite of causing an approxi
mately
75% reduction
in replication,
ADM
(1 @.tg/ml) did not
inhibit UV-stimulated repair synthesis (Chart 2; Table 1).
However, at higher doses of ADM (2, 5@and 10 j@g/ml),
inhibition of UV-induced DNA repair was observed (Table
1). Supportive
evidence of such inhibition
is seen in recent
studies on human leukocytes by Ringborg and Lambert
(30). Comparable
results have also been found by Byfield et
a!. (7) and Lee et a!. (24) who showed that X-ray-induced
excision repair is somewhat limited in mammalian tumor
cells treated with and maintained in the presence of ADM.
To account for the differential action of ADM on DNA
replication and UV-induced DNA repair in cardiac cell
populations, we suggest that the focus of drug action may
be limited to a site or sites proximal to where the drug
intercalates into the double helix. Perhaps only one or a
few ADM molecules positioned in the path of an oncoming
CANCER
RESEARCH
VOL. 39
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Effect of ADM on DNA Replication and Repair
Effect of UV irradiation
wasadded
Effect of UV irradiation
[3H]dThdpulse
at a concentration
Table 1
on replicative
and repair synthesis
on replicative and repair synthesis in control and ADM-treated cardiac cultures. ADM
of 1 , 2, 5, or 10 @g/mlat 18 to 21 hr. UV irradiation at 68 hr was followed by a 4-hr
expressedas
in the presenceand absenceof 0.1 M HU.The data represent UV-stimulated[3H)dThdincorporation
a percentageof the control value (no UV present)for
sample.UV
each
mm)0
irradiation
675[3H]dThd
intensity
(ergs/sq
15
150
in
corporationb(cpm/@tgprotionbHG
Conditions
N@'
tein)
N
%stimulation@@
%stimulationb
N
%stimula
N
2HG
11
317 ±42@
9
75 ± 6
14
31 ± 6
14
12 ±
12ADM1@'
+ HU
11
11 ± 2
9
103 ± 5
16
142 ±13
16
142 ±
8ADM1+HU
178±24ADM2
9ADM2+HU
11
11
6
75±16
5± 1
54±11
9
9
5
83± 5
137± 6
94± 7
11
11
6
39± 4
146±20
40± 5
10
11
6
20±
106±10ADM5
7
4± 1
5
106±15
7
137±18
8
9ADM5+HU
6
38± 7
5
94±13
6
45± 8
5
114±20ADM1O
7
5
150±32
7
106±22
7
72±20ADM1O+HU
7
7
5
5
85±13
89±13
7
7
56±16
90±15
7
7
4, 6, and
8) is calculated
3± 0.8
19±
3±
3
0.5
18±
44±
76±18
aN,number
ofexperiments.
b Percentage
of
corresponding
(.
Mean
stimulation
unirradiated
of
the
UV-irradiated
sample (Column
samples
following
ADM,
concentration
of the drug
designating
each
(@g/ml).
replication fork might be sufficient to impede further prog
messof the fork beyond the drug-binding site. Thus replica
tion could be inhibited at relatively low ADM concentra
tions.
This, however, would not be the case for repair synthesis.
The production of UV-induced pyrimidine dimers elicits an
excision-repair response. If the ADM were administered at
a high enough concentration for its molecules to be located
within about 50 nucleotide pairs of a dimem (13), DNA
polymerase could be physically inhibited from repairing the
gapped region. It is not currently feasible to quantitate the
number of intercalated ADM molecules per pyrimidine di
mer in cardiac cultures. However, it can be reasonably
hypothesized that (a) a drug:dimer ratio exists at which
replication is decreased while UV-stimulated repair is unaf
fected, and (b) at some higher ratio, both forms of synthesis
are reduced.
An alternate model might be proposed since a direct
physical impediment by ADM may not necessarily be re
quired to inhibit DNA synthesis. In fact, a precedent for
indirect physical interference or ‘
‘action
at a distance' ‘
has
been noted in biological systems in vitro by Burd et a!. (3).
They observed that a perturbation at a localized DNA region
can affect the stability of a distant DNA region. Intercalating
agents such as ADM which cause unwinding of DNA are
excellent candidates for exhibiting biological effects over
regions beyond their discrete binding sites. Our data are
more compatible with the previous model in which ADM
must intercalate near the dimer region to interfere with
repair synthesis.
Since ADM has been observed in numerous instances to
cause DNA breakdown in vivo (5, 7, 31), it was of interest to
whether
by
± SE.
d Numbers
inquire
(Columns
2) as 100% [3H]dThd incorporation.
ADM
could
itself
stimulate
DNA
that ADM-induced DNA degradation could evolve from the
abortive nicking action by endonucleases attempting to
free the DNA of its tightly bound drug contaminant. Our
results (Table 1) indicate that in the presence of HU,
unirradiated
ADM-treatedcellsdo not stimulate[3H]dThd
incorporation
overunirradiated
control(HG plusHU) levels.
The inhibition of unscheduled DNA synthesis in cultured
cardiac cells by ADM at 10 @g/mlbut not at 1 @zg/mland an
observed ADM-related fluorescence in the cells 2 days after
treatment suggest a plausible mechanism for ADM cardi
otoxicity. The toxicity of ADM when given in repeated doses
in chemotherapy may result from an accumulation of the
drug bound to DNA to the extent that repair of drug-induced
damage can no longer occur.
REFERENCES
1. Blake, A. , and Peacocke, A. R. The interaction of aminoacridines with
nucleic acids. Biopolymers, 6: 1225—1253,
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Fig. 1. Autoradiographs of cultured rat heterogeneous cardiac cells undergoing DNA-replicative or repair synthesis. ADM was added at a concentration of
I , 2, 5, or 10 @g/mlat 18 to 21 hr of culture growth. At 68 hr, UV irradiation at 675 ergs/sq mm was followed by a 4-hr pulse of @H]dThd(10 @.iCi/ml)in the
presence or absence of 0.1 M HU. Arrows, labeled nuclei. A, unirradiated HC; B, irradiated HC plus HU; C, unirradiated ADM (1 @g/ml);D, irradiated ADM
plus HU (1 @tg/ml);E, unirradiated ADM (2 @tg/ml);F, irradiated ADM plus HU (2 .tg/ml); G, unirradiated ADM (5 @tg/ml);H, irradiated ADM plus HU (5 .tg/ml);
I, unirradiatedADM(10 @g/ml);J,
irradiatedADMplus HU(10@tg/ml).
1326
CANCER RESEARCH VOL. 39
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Differential Effect of Adriamycin on DNA Replicative and Repair
Synthesis in Cultured Neonatal Rat Cardiac Cells
Henie Fialkoff, Myron F. Goodman and Maria W. Seraydarian
Cancer Res 1979;39:1321-1327.
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