Download Effects of Adriamycin on Supercoiled DMA and

[CANCER RESEARCH 44, 613-618,
February 1984]
Effects of Adriamycin on Supercoiled DMA and Calf Thymus Nucleosomes
Studied with Fluorescent Probes1
Henry Simpkins,2 Leslie F. Pearlman, and Leslie M. Thompson
Departments of Pathology and Biological Chemistry, California College of Medicine, University of California, Irvine, California 92717
ABSTRACT
The interaction of the antitumor drug Adriamycin with nucleotides, polynucleotides, RNA, calf thymus nucleosomes, and DMA
(including pBR322 supercoiled DMA) has been studied using
fluorescent probes. The lanthanide terbium is known to interact
with guanine and xanthosine to produce high fluorescence en
hancement. The nature of the interaction of the lanthanide with
the heterocyclic ring in guanine appears to involve both the C-2
and N-7 groups. A striking decrease in fluorescence enhance
ment was observed with all of the polynucleotides, RNA, DNA,
and nucleosomes after treatment with Adriamycin at molar ratios
of 1:200 or less. It appears that Adriamycin interacts with the
guanine ring, displacing or preventing terbium access to its
second site of binding. However, with supercoiled DNA and
nucleosomes, the displacement followed a destabilization of the
helix at very low drug concentrations. The binding affinities of
calf thymus DNA, pBR322 DNA, and calf thymus nucleosomes
at 37° for Adriamycin were of the same order of magnitude.
Reaction with A/-pyrene maleimide, a fluorescent probe which
binds to histone H3, showed that Adriamycin interacted with the
nucleosome to increase the binding of the probe (only, however,
at drug ratios far greater than those required to produce effects
with DNA). No compositional changes of supercoiled or nucleosomal DNA or nucleosomal histones were observed by agarose
gel or sodium dodecyl sulfate:polyacrylamide gel electrophoresis,
respectively. The classic intercalating agent, ethidium bromide,
produced minimal displacement of the lanthanide from DNA,
although an effect with RNA at high drug concentrations was
observed.
INTRODUCTION
Adriamycin is an anthracycline antibiotic currently used as a
potent chemotherapeutic agent in the treatment of certain leukemias and solid tumors (2). The primary target site of this drug
has generally been accepted to be nuclear DNA. Adriamycin has
been assumed to intercalate into native DNA (9) and, when
added to cells in culture, it is known to inhibit both DNA and
RNA synthesis (6, 20). The physicochemical effects of the drug
on isolated DNA have generally been examined at very high
drug:DNA ratios (22).
Adriamycin has recently been shown to produce single-strand
breaks when cells are treated in vivo, although it has been
suggested that some of the breaks are really of a doublestranded nature. It has been postulated that a topoisomerase
binds to the terminus of one of the strands (23, 26). Since some
1 Financial support was received from the Department of Pathology, California
College of Medicine, University of California, Irvine, Irvine, CA.
2To whom requests for reprints should be addressed.
Received March 29,1983; accepted November 8,1983.
FEBRUARY
1984
derivatives of Adriamycin do not bind to DNA but are cytotoxic
(16), the necessity for a priori binding has to be questioned. It
has also been shown that Adriamycin is reduced by microsomal
enzymes to semiquinone-free radicals which do not have to
intercalate into DNA to produce strand breakage (1, 26). With
the exception of 2 recent articles, very little has been reported
of the interaction of this drug with nuclei or nuclear chromatin.
Adriamycin has been shown to produce single-stranded DNA
breaks in isolated nuclei, as determined by the sensitivity of the
treated nuclei to Neurospora crassa nuclease (3). Electron mi
croscopy as well as the analysis by alkaline sucrose gradients
of DNA from the treated chromatin has shown morphological
and compositional changes (33). These effects, however, were
not observed if the nuclear proteins in the chromatin were
hydrolyzed by proteases prior to drug treatment. This report
prompted us to examine the effects of Adriamycin on DNA and
protein structure within the nucleosome utilizing 2 highly sensi
tive and specific fluorescent probes, terbium and A/-pyrene mal
eimide.
Terbium produces strong fluorescent enhancement when
bound to guanine and xanthosine bases when they are in singlestranded structures (12, 29). Since it has been proposed that
Adriamycin acts primarily on G-C sequences (7), terbium is a
potentially powerful tool for examining the drug-DNA interaction
within the nucleosome. A/-pyrene maleimidfc reacts with thymocyte and rat liver chromatin in situ, labeling the cysteine groups
in histone H3 (probably cysteine 96), thus providing a means of
monitoring the conformation of the histone octamer in the chro
matin strand. We have used this probe to show conformational
changes in chromatin induced by salt (21), liver regeneration
(25). and the antitumor drug c/s-dichlorodiammineplatinum(ll)
(28). In addition, the intrinsic fluorescence of Adriamycin has
been used to monitor its binding to DNA and polynucleotides (7,
37). We have continued these observations using calf thymus
mononucleosomes.
MATERIALS AND METHODS
Reagents. Adriamycin was obtained from Sigma Chemical Company
and was used without further purification. Commercially prepared nucleic
acids were obtained from Sigma Chemical Company, P-L Biochemicals,
Miles Biochemicals, and Boehringer-Mannheim. They were suspended
directly into Tris-HCI, pH 7.4, at a final concentration of 1.00 mw
nucleotide phosphate, as determined by the respective extinction coef
ficients. Identical results were obtained with DNA which had been further
purified with RNase T2 digestion followed by chloroformiisoamyl alcohol
extraction. Supercoiled DNA from plasmid pBR322 was prepared by
amplification in the presence of chloramphenicol according to the method
of Clewell (5), and the DNA was purified by the clear lysate technique of
Guerryefa/. (13).
Preparation of Nucleosomes. Calf thymus nucleosomes were pre
pared using a modification of the procedure described by Levy-Wilson
ef al. (17). Calf thymus (5 g) was homogenized
in Buffer A [0.25 M
613
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
H. Simpkins et al.
sucrose:10mw
MgCI2:1 mw CaCI2; 1 mM PMSF3:0.1% (w/v) Triton X-
100:50 mM Tris-HCI, pH 7.4] then washed 3 times ¡nBuffer A without
Triton X-100. Nuclei were suspended in Buffer B (10 mM NaCI:3 mw
MgCI2:1 mM PMSF:10 mM Tris-HCI, pH 7.5) and then washed twice in
the same buffer containing 1 mM CaCI2 and digested for 30 min with
micrococcal nuclease (Worthington Biochemicals, 25 units/ml) at a DMA
concentration of 10 mg/ml. The reaction was terminated by chilling the
tubes and centrifuging for 5 min at 4050 x g in a Sorvall HB-4 rotor. The
pellet was resuspended in Buffer C (1 mM EDTA:1 mM PMSF:10 mM
Tris-HCI, pH 7.5), swelled on ice for 20 min, and centrifuged for 20 min
at 16,300 x g. The last step was repeated, the 2 supematants were
combined, and the nucleosomes were recovered by precipitation with 3
volumes of ethanol in 0.1 M NaCI. After centrifugation, the nucleosomes
were suspended in storage buffer (0.2 mM EDTA:1 mM PMSF:10 mM
Tris-HCI, pH 8.0) and kept frozen at -20° until needed for use. These
nucleosomes possessed a full complement of histones, including H1,
when monitored by SDS gel electrophoresis.
Terbium Fluorescence Measurements. Samples containing the ap
propriate concentrations of nucleic acid and terbium were made up to
1.2 ml in 20 mM Tris-HCI, pH 7.4. The samples were equilibrated for 30
min at room temperature, by which time the reaction was determined to
be complete. Experiments involving RNA were equilibrated at 4°for 30
min and then brought to room temperature, and fluorescence was
measured immediately, since lanthanides have been shown to hydrolyze
the nucleotide bonds in RNA, although slowly, at room temperature (8).
The fluorescence intensity was measured using 1.0-cm quartz cells, in a
Perkin Elmer MPF 43A Fluorescence Spectrophotometer equipped with
a high-pressure xenon lamp. An excitation wavelength of 290 nm and
an emission wavelength of 544 nm were optimal for maximizing energy
transfer and minimizing background light scattering. Entrance and exit
slit widths were maintained at 10 nm for all fluorescence measurements.
The concentration of terbium chloride-6H2O (Aldrich Chemicals) was
made up to 100 mM in distilled water and precisely determined by titration
against EDTA (12).
Titrations with the terbium ion were performed on samples containing
identical quantities of the nucleic acid by the addition of concentrated
terbium chloride (1 or 10 mM).
Titration of the Nucleosomes with At-Pyrene Maleimide. N-pyrene
maleimide was synthesized from 3-aminopyrene as described by Weltman ef al. (34). Prior to use, it was dissolved as a 0.1 mM solution in
ethanol. The nucleosomes [20 ^g of protein as determined by the method
of Lowry ef al. (18) using total calf thymus histone as standard] were
incubated with increasing concentrations of Adriamycin for 20 min at 37°
in the nucleosomal storage buffer. The treated nucleosomes were then
precipitated with 3 volumes of ethanol in 0.1 M NaCI, pelleted by
centrifugation for 15 min at 3000 x g, resuspended in storage buffer,
and reacted with 0 to 200 nM A/-pyrene maleimide for 25 min at room
temperature. The fluorescence intensity was measured using an excita
tion wavelength of 342 nm and an emission wavelength of 377 nm.
Similar results were obtained if the treated nucleosomes were recovered
by centrifugation at 400,000 x g for 4 hr in lieu of ethanol precipitation
and then labeled with N-pyrene maleimide.
Treated and control nucleosomes (0.5 mg) were incubated with trypsin
(0.1 mg) for 60 min at 37°.DNA fragments were subsequently extracted
using a 10-fold excess of chlorofornvisoamyl
alcohol (24:1, v/v). After
centrifugation for 10 min at 3000 x g, the aqueous layer was carefully
removed, and the DNA precipitated with 3 volumes of ethanol in 0.1 M
NaCI. The DNA (5 Mg) was analyzed on agarose gels according to the
procedure of McMaster ef al. (19). The gels were stained with ethidium
bromide (1 Mg/ml) tor 30 min, destained overnight at 4°with distilled
water, and then photographed using Polaroid type 55 film, utilizing a 63B
Transilluminator (Ultra-Violet Products, San Gabriel, CA).
Thermal Melting Profiles. Adriamycin-treated and control DNA and
nucleosomes were made up to 1.0 mM DNA phosphate in 10 mM sodium
phosphate buffer, pH 7.4, with 0.2 mM EDTA and 1 mM PMSF, and
diluted in the same buffer to give an absorbance at 260 nm of 0.6 to 0.8.
The samples were subjected to an increase in temperature at a rate of
1°/minusing a Haake FJ thermal circulating pump. The absorbance was
read at 260 nm on a single-beam Gilford 250 Spectrophotometer
using
buffer as the blank.
RESULTS
When DNA was treated with Adriamycin at a 1:20 or 1:50 (w/
w) ratio for 20 min at 37°and precipitated with ethanol in 0.1 M
NaCI and when terbium binding curves were performed, a
marked dimnuition in the terbium fluorescence was observed,
almost to within 25% of that observed with the nontreated
samples. The reaction appears complete by this time, because
similar drug concentrations at 10, 30, and 60 min of incubation
produced the same decreases in fluorescence. We documented
this decrease with calf thymus DNA and a mixture of 16S and
23S fragments of Escherichia coli rRNA by treatment with in
creasing concentrations of Adriamycin at 100 pM terbium (Chart
1). Both nucleic acids, with markedly different secondary struc
tures (a double-stranded molecule versus one containing singlestranded regions), show a substantial decrease in terbium fluo
rescence, the intensity reaching a plateau at approximately 1 to
1.5 pM Adriamycin. The decrease in fluorescence intensity ap
pears at lower drug concentrations with the less structured
molecule.
When the guanine-containing homoplymers poly(rG) and
poly(dG) were titrated with Adriamycin, the polyribonucleotide
showed a greater decrease than did the deoxyribose derivative
lOO.t
Compositional Studies. The proteins of the treated and control
nucleosomes were analyzed by SDS polyacrylamide gel electrophoresis
using a modification of the procedure of Laemmli (15). The stacking gel
contained 4.2% (w/v) acrylamide with a bisacrylamide:acrylamide ratio
of 1:37.5 (w/w) in 0.1% SDS:0.125 M Tris-HCI, pH 6.8; the separating
gel contained 12% (w/v) acrylamide in 0.1% SDS:0.375 M Tris-HCI, pH
8.8; and a reservoir buffer contained 0.1% SDS:0.19 M glycine:0.125 M
Tris-HCI, pH 6.8. The current was kept constant at 7.5 mA until the
samples had run through the stacking gel and then at 12.5 mA until the
tracking dye was within 0.5 cm of the bottom. The gels were stained
with 0.1% (w/v) Coomassie Blue in methanokacetic acid:water (10:7:83,
v/v) and destained with the same methanol:acetic acid:water mixture.
3 The abbreviations used are: PMSF, phenylmethylsulfonylfluoride; SDS, sodium
dodecyl sulfate; poly(rG). polynboguanylate; poly(dG), polydeoxyguanylate.
614
02 03 04 0.5 06 07 08
Adriamycin
09
10
Chart 1. Terbium fluorescence (F) expressed as a percentage of the control
(Fc). Increasing concentrations (0 to 4 MM)of Adriamycin were incubated with 50
MMcalf thymus DNA (•)or 50 MM 16S and 23S RNA (A) at 37°for 20 min in 20
mM Tris-HCI, pH 7.4. The terbium was then added at a constant concentration of
100 MM.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
VOL. 44
Effects ofAdriamycin
on DNA and Nucleosomes
(Chart 2). The decrease in fluorescence intensity of poly(rG)
occurred at 0.5 pM Adriamycin as compared to 1 U.M with
poly(dG). These polymers are essentially nonstructured at this
concentration and ionic strength, although poly(dG) forms a 4stranded helix at higher concentrations (35). Since it is known
that terbium fluoresces more intensely when reacted with guanine residues in a single-stranded random coil than in a doublestranded helical form, it was important to determine whether this
result was due to single-stranded breaks or to nicks in the linear
DNA fragments.
To this end, the titration of Adriamycin with supercoiled plasmid pBR322 DNA was performed (Chart 3). A similar decrease
in overall fluorescence was found. Fluorescence intensity plateaued at 1 to 1.5 ^M Adriamycin, as with calf thymus DNA and
poly(dG). A marked increase in fluorescence was observed at
low Adriamycin concentrations (0.05 to 0.2 ¿IM).
This effect was
not apparent with linear DNA fragments from calf thymus, nor
did it occur when the drug was added to a terbium:nucleic acid
mixture at 25°and the titration was performed directly, where
the shape of the curve is more hyperbolic. A curve similar to that
generated with the pBR322 DNA was obtained when calf thymus
nucleosomes were titrated with the drug.
In order to determine whether the observed decreases in
terbium fluorescence were caused by an intrinsic effect on the
guanine residues themselves, the nucleotides GMP, XMP, and
several derivatives of guanine were titrated (at far higher con
centrations, since their intrinsic fluorescence with the lanthanide
is so much lower). A marked decrease in terbium fluorescence
was observed (Table 1).
The possibility that the drug, which exhibits minimal fluores
cence at 544 nm when excited at 290 nm, quenches terbium
fluorescence was considered. However, the fluorescence of
Adriamycin is only 5 to 10% of that observed with terbium-DNA
complexes at a 1 to 2 MMdrug concentration (highest concentra
tion used) and less than 1% for polynucleotide:terbium com
plexes (due to the higher intrinsic fluorescence of these com
plexes with terbium). In addition, when the DNA or polynucleotide
was treated with Adriamycin, precipitated, and resuspended
(thereby removing any unbound Adriamycin) and a terbium titra
tion curve was performed, values similar to our previous data
were obtained.
0
01 02 03 04 05 0.6 07 08 09
10 I.I
pM Adriamycin
Chart 3. Terbium fluorescence(F)expressed as a percentageof the control (Fc)
of a 50 fiu solution of supercoiledp8R322 DNA to which increasingconcentrations
of Adriamycin had been added at 37°for 20 min in 20 mw Tris-HCI, pH 7.4, prior
to the terbium (O). G, fluorescence of a solution of 50 /JMpBR322 DNA reacted
with 100 liM terbium at 25°,to which Adriamycin was added at the concentrations
indicated. A, effect of treatment for 20 min at 37°with increasing concentrations
of Adriamycin on the terbium fluorescence of calf thymus nucleosomes in 10 mm
triethanolamineHCI, pH 7.4.
Table 1
Effect ofAdriamycin on terbium fluorescenceof guanine nucleotides
The nudeotide and drug were incubated at the concentrations described below
for 20 min at 37°in 20 mw Tris-HCI, pH 7.4, prior to the addition of the terbium.
Data are expressed as a percentage of the fluorescenceof the control, which was
incubated with buffer substituted for the Adriamycin.
inten
sity at 100 pu ter
bium10062
NucleotidePoWrG)5--GMP5'-XMP7-Methyl-5'-GMPNucleotide50
(Õ.M)0
(2)"27
+ 5a
0.100.4800.311.5300.271.3600.180.90Fluorescence
5050200200200200200200200200200Adriamycin
(2)10063
+3
(2)17
+7
(2)10053
+2
(2)13
+5
(2)10080
+2
(2)45
+7
+ 5 (2)
* Mean ±S.E.
" Numbers in parentheses, number of determinations.
02 0.3 04 0.5 0.6 07 0.8 09 1.0
/U.MAdriamycin
Chart 2. Terbium fluorescence (F) expressed as a percentage of the control
(Fc). Solutions (25 pM)of the polynucleotidespoly(dG)(•)
or poly(rG)(A) to which
increasing concentrations of Adriamycin had been added at 37°for 20 min in 20
mMTris-HCI, pH 7.4, are shown. Terbium was then added at a constant concen
tration of 100 MM
FEBRUARY
1984
When the effect of low concentrations of the drug was ana
lyzed by thermal melting of the Adriamycin:DNA complexes (1:50
or greater molar ratio), it was found that the effect was minimal,
a stabilization of the order of 1-2° in the melting temperature
(Tm).This was true whether isolated linear DNA fragments from
E. coli, calf thymus, or calf thymus nucleosomes were used. The
15-16° stabilization in Tm (22) at high drug:DNA (1:10) ratios
was not observed at these relatively low concentrations
short incubation times.
and
615
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
H. Simpkins et al.
The effect of a classic intercalating agent, ethidium bromide,
on terbium displacement was investigated. It was found that this
drug caused no appreciable decrease in the terbium fluorescence
of calf thymus DNA but did diminish, although at far higher drug
concentrations, the fluorescence of rRNA from E. coli (Chart 4).
The effect of Adriamycin on nucleic acid:protein complexes
was investigated by reacting calf thymus nucleosomes with the
drug. The treated nucleosomes were pelleted, the terbium bind
ing curves were performed in 10 mM triethanolamine-HCI, pH 7.4
(Chart 5). It is observed that, in contrast to the curves obtained
with polynucleotides and isolated DNA molecules, saturation by
terbium was not observed even at 700 tiM and that an almost
linear increase in fluorescence was observed with increasing
terbium concentrations. Terbium fluorescence, however, was
still markedly decreased by prior treatment of the nucleosomes
130
120
110
100
with findings of other authors (7, 37), decreases in fluorescence
were observed which could be analyzed by a Scatchard analysis
(See Table 2). Nucleosomes and calf thymus DNA showed similar
affinities and number of binding sites for the drug. Supercoiled
DNA showed a somewhat lower number of binding sites, al
though the affinity constants were of the same order of magni
tude. The values obtained with calf thymus DNA are of the same
order of magnitude, as reported by other authors at lower
temperatures and higher ionic strengths (7, 37). Minimal effects
of temperature were observed (data not presented).
To analyze the effect on the histone protein conformation
within the nucleosome, the fluorescent probe A/-pyrene maleimide was used. This probe has been shown by us (21, 25) to label
histone H3 in situ within the calf thymus or rat liver nucleosome,
probably at the cysteine 96 residue. When the binding of A/pyrene maleimide was analyzed subsequent to Adriamycin treat
ment of calf thymus nucleosomes, it was found that an increase
in W-pyrene maleimide fluorescence was observed only at very
high drug concentrations (Table 3). When the proteins of the
treated nucleosomes were analyzed by polyacrylamide gel electrophoresis, no changes in protein composition were observed,
even at high Adriamycin concentrations (data not presented). In
addition, when calf thymus DNA, pBR322 DNA, or calf thymus
90
80
B 70
£60
-50
40
30
20
10 h
0
with increasing Adriamycin concentrations, even at a Adriamycin:DNA molar ratio as low as 1:200.
Titration of Adhamycin with calf thymus DNA, pBR322 DNA,
and calf thymus nucleosomes was performed at 37°.In accord
O.I 0.2 0.3 0.4 05 06 07 08 0.9 1.0
fiM Ethidium Bromide
Chart 4. Terbium fluorescence (F) expressed as a percentage of the controls
(Fc). Calf thymus DNA (50 JIM) (A) and 16S and 23S rRNA (O) treated with
increasing concentrations of ethidium bromide in 20 mu Tris-HCI, pH 7.4, at 37°
for 20 min, followed by the addition of 100 /IM terbium, are shown.
Table 2
Association constants and the number of binding sites for Adriamycin binding to
RNA, DNA. and calf thymus nucleosomes
The drug was kept at a constant concentration of 15 MM, and the DNA-P
concentration was increased until fluorescence intensity leveled. The buffer used
was 10 mM sodium phosphate, pH 7.4, unless otherwise noted. Experiments were
performed at 37°.An excitation wavelength of 480 nm and an emission wavelength
of 560 nm were used. The curves were analyzed as described by Scatchard (24).
acidCalf
no. of
binding
sites0.28
Nucleic
thymus DNA
(10 mM Tris-HCI:0.2 mM EDTA:1
[20
2(2)]20
±
mM PMSF, pH 7.4)
±3(2)
pBR322 DNA
(10 mM Tris-HCI:0.2 mM EDTA:1
[19
±3(2)]10
mM PMSF, pH 7.4)
±2(2)Apparent
Calf thymus nucleosomes
(10 mM Tris-HCI:0.2 mM EDTA:1
mM PMSF, pH 8.0)Ka(x10>)M-1'i10±2"(2f
" Association constant.
6 Mean ±S.E.
c Numbers in parentheses, number of determinations.
±0.05
[0.25
0.05]0.21
±
±0.04
[0.19
0.03]0.30
+
±0.05
Table 3
Effecf of Adriamycin on N-pyrene maleimide fluorescence of calf thymus
nucleosomes
Calf thymus nucleosomes were incubated with Adriamycin at the indicated ratios
for 20 min at 37°in 10 mw Tris-HCI, pH 8.0, with 0.2 mM EDTA and 1 mM PMSF,
precipitated with ethanol, and resuspended in the same buffer prior to the addition
of N-pyrene maleimide. The data are expressed as a percentage of the control
fluorescence, which was incubated with buffer substituted for the Adriamycin.
800
Chart 5. Calf thymus nucleosomes were reacted with increasing molar ratios of
Adriamycin for 20 min at 37°. O, no drug; A, 1:200 (drug:DNA-P); D, 1:20
(drug:DNA-P). The nucleosomes were pelleted following drug treatment and resuspended in 10 mM triethanolamine-HCI, pH 7.4. Binding curves were performed by
increasing the concentration of terbium at 50 ¿IM
DNA-P.
616
Concentra
tions of Npyrene mal
eimidenM20
ratios1:10150
intensities at various drug:nucleosomal
(w/w)
protein
±15a
+ 15
±1585
±15
±15
130 ±15
200 ±20
185 ±20
50
110± 15
95 ±15
240 ±20
170 ±20
100200Fluorescence
135 ±15
100 ±151:500100
150 ±151:20150 190 ±201:200120
a Mean ±S.E.
CANCER RESEARCH
VOL. 44
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
Effects of Adriamycin on DNA and Nucleosomes
nucleosomes were treated with varying concentrations of Adri
amycin and the DNA was analyzed by agarose gel electrophoresis, no reproducible difference in electrophoretic mobility was
observed in any of the samples. This suggests that modification
of the DNA or strand breakage of the supercoiled DNA had not
occurred.
DISCUSSION
The mode of action of Adriamycin has become a matter of
discussion, in view of the recent reports showing that it produces
single- and double-strand breaks in DNA in vivo and that a priori
binding to the DNA may not be necessary for this reaction to
occur (16, 23, 36). In addition, recent reports that the drug is
reduced by microsomal enzymes to free radicals opens up new
avenues of research (1, 26), as do the many reports showing
effects at the membrane level (10, 27, 30, 31). A recent article
(32) which shows that the drug, when coupled to agarose (which
renders it impermeable to cells), results in a product still markedly
cytotoxic to L1210 leukemic cells, raises more important ques
tions as to its mode of action. It is obvious that a simple
intercalation model with DNA cannot totally explain its mecha
nism of action. This idea is strengthened by our results, which
show that displacement of terbium from DNA produced by this
drug is not duplicated by ethidium bromide. However, it should
be pointed out that ethidium produces a much greater unwinding
of DNA than does Adriamycin (26°as opposed to 10-13°); thus,
the mode of intercalation may be different.
We have concluded that Adriamycin reacts with guanine resi
dues, somehow distorting the ring such that the intrinsic fluores
cence of the ion which requires 2-site binding (12) does not
occur with either nucleotide, polynucleotide, RNA, singlestranded DNA, supercoiled DNA, or the intact nucleosome. It is
noteworthy that the effects are observed at relatively low Adria
mycin concentrations, suggesting a very high specificity and
affinity of the drug for guanine residues. This may be biologically
relevant.
This interaction is very interesting when compared to results
obtained with displacement studies using cations (14). It was
found that Cu(ll), which is known to interact with both the
phosphate group and the base ring (11), was the most effective
counterion to terbium, having twice the affinity exhibited by
terbium itself. Adriamycin produces its effect at 0.5 to 1.0 ^M
with 50 ftM nucleotide, showing approximately a 10- to 20-fold
increase in affinity over the cuprous ion. This may be due to both
its interaction with the base ring and its specificity for guanine
bases, which is not apparent with the cuprous ion. The Km for
terbium for supercoiled DNA is approximately 105 M"1 (12), and
we and others (7, 37) report values of 107 w~1 for the affinity of
Adriamycin for DNA and calf thymus nucleosomes. However, it
is possible that the reaction of the drug with A-T regions proximal
to guanine residues may result in the observed change. This
cannot be discounted, in view of the result of Chandra ef al. (4)
showing drug-induced inhibition of DNA polymerase activity with
A-T polynucleotide templates.
The initial destabilization of the helix at low drug concentrations
observed with supercoiled pBR 322 DNA and nucleosomes prior
to the displacement of terbium from its second binding site (12)
may also be biologically relevant. This results in a marked in
crease in fluorescence intensity. This effect on terbium fluores
cence has also been observed when supercoiled DNA is titrated
FEBRUARY
1984
with low salt concentrations (11).
Finally, the drug appears to have little effect on nucleosomal
histones as monitored by A/-pyrene maleimide binding studies.
This probe was used successfully (28) to show marked effects
of the potent antitumor drug c/'s-dichlorodiammineplatinum(ll) on
nucleosomal proteins at very low drug concentrations and short
incubation times, conditions under which no changes in DNA
conformation could be detected. Conventional polyacrylamide
gel electrophoresis also showed no detectable effect on nuclear
proteins following reaction with Adriamycin, although marked
changes in terbium fluorescence of the nucleosomes treated
with very low concentrations were observed.
Thus, the drug appears to react in vitro at very low concentra
tions with guanine residues, perturbing the interaction of terbium
with the guanine ring so that the fluorescence enhancement of
the lanthanide produced by this base is abrogated. This effect is
also observed with supercoiled DNA as well as with protein:DNA
complexes, i.e., nucleosomes. However, the classic intercalating
agent, ethidium bromide, did not produce any effect with DNA.
ACKNOWLEDGMENTS
The authors wish to thank Luis de la Maza, M.D., Ph.D., and Marie Pollack for
the generous gift of pBR 322 supercoiled DNA and Rachel Hwei-Ping Yu for her
technical assistance.
REFERENCES
1. Berlin, V., and Haseltine, W. A. Reduction of Adriamycin to a semiquinone-free
radical by NADPH-cytochrome P450 reducÃ-ase produces DNA cleavage in a
reaction mediated by molecular oxygen. J. Biol. Chem., 256: 4747-4756,
1981.
2. Blum, R. H., and Carter, S. K. Adriamycin. A new anticancer drug with
significant clinical activity. Ann. Intern. Med., 80: 249-259,1979.
3. Center, M. S. Induction of single stranded regions in nuclear DNA by Adria
mycin. Biochem. Biophys. Res. Commun., 89: 1231-1238,1979.
4. Chandra, P., Zunino, F., Gotz, A., Gericke, D., and Thorbeck, R. Specific
inhibition of DNA-polymerases from RNA tumor viruses by some new daunomycin derivatives. FEBS Lett., 27: 264-268, 1972.
5. Clewell, D. B. Nature of col E plasmid replication in Escherichia coli in the
presence of chloramphenicol. J. Bacteriol., 770: 667-676,1972.
6. Crooke, S. T., Duvemay, V. H., Galvan, L., and Prestayko, A. W. Structureactivity relationship of anthracyclines relative to effects on macromolecular
syntheses. Mol. Pharmacol., 74:290-298,1978.
7. Duvemay, V. H., Pachter, J. A., and Crooke, T. S. Deoxyribonucleic acid
binding studies on several new anthracyclic antitumor antibiotics. Sequence
preference and structure activity relationship of marcellomycin and its ana
logues as compared to Adriamycin. Biochemistry, 18: 4024-4030,1979.
8. Eichhorn, G. L., and Butzow, J. J. Interaction of metal ions with polynucleotides
and related compounds. III. Degradation of polyribonucleotides by lanthanum
ions. Biopolymers, 3: 79-94, 1965.
9. Gabbay, E. J., Grier, D., Fingerle, R. E., Reimer, R., Levy, R., Pearce, S. W.,
and Wilson, W. D. Interaction specificity of the anthracyclines with deoxyribonucleic acid. Biochemistry, 75: 2062-2070,1976.
10. Goormaghtigh, E., Châtelain, P., Caspers, J., Ruysschaert, J. M. Adriamycin
inhibits the formation of non-bilayer lipid structures in cardiolipin-containing
model membranes. Biochim. Biophys. Acta, 597:1-14,1980.
11. Gross. D. S., Rice, S. W., and Simpkins, H. Influence of inorganic cations and
histone proteins on the terbium(lll)-nucleic acid interaction. Biochim. Biophys.
Acta, 656: 167-176,1981.
12. Gross, D. S., and Simpkins, H. Evidence for two site binding in the terbium(lll)nucleic acid interaction. J. Bid. Chem., 256: 9593-9598, 1981.
13. Guerry, P., LeBlanc, D. J., and Falkow, S. General method for the isolation of
plasmid deoxyribonucleic add. J. Bacteriol., 776:1064-1066,1973.
14. Izatt, R. M., Christensen, J. J., and Rytting, J. H. Sites and thermodynamic
quantities associated with protons and metal ion interactions with ribonucleic
acid and deoxyribonucleic acid and their constituent bases, nudeosides and
nucleotides. Chem. Rev., 77: 439-481, 1971.
15. Laemmli, V. K. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature (Lond.), 227. 680-685,1971.
16. Levin, M., Silber, R., Israel, M., Goldfeder, A., Khetarpal, V. K., and Potmesil,
M. Protein-associated DNA breaks and DNA-protein cross-links caused by
DNA nonbinding derivatives of Adriamycin in L1210 cells. Cancer Res., 47:
1006-1010,1981.
17. Levy-Wilson, B., Connor, W., and Dixon, G. H. A subset of trout testis
nucleosomes enriched in transcribed DNA sequences containing high mobility
617
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
H. Simpkins et al.
group proteins as major structural components. J. Biol. Chem., 254:609-620,
1979.
18. Lowry, 0. H., Rosebrough, N. J., Fair, A. L, and Randall, R. J. Protein
measurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275,
1951.
19. McMaster, G. K., and Carmichael, G. C. Analysis of single and double stranded
nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine
orange. Proc. Nati. Acad. Sei. U. S. A„74: 4835-4838,1977.
20. Meriwether, W. D., and Bachur, N. R. Inhibition of DNA and RNA metabolism
by duanorubicin and Adriamycin in U 210 mouse leukemia. Cancer Res., 32:
1137-1142.1972.
21. Mooney, D., Thompson, L. M., and Simpkins, H. The in situ labeling of historie
H-3 in chromatin by a fluorescent probe. Biochim. Biophys. Acta, 625: 51-63,
1980.
22. Neidle, S. Daunomycin and related compounds. Prog. Med. Chem., ?6: 196221,1979.
23. Ross, W. E., and Bradley, M. 0. DNA double-stranded breaks in mammalian
cells after exposure to interacalating agents. Biochim. Biophys. Acta, 654:
129-134, 1981.
24. Scatchard, A. The attraction of proteins for small molecules and ions. Ann. N.
Y. Acad. Sci., 5T: 660-672, 1949.
25. Simpkins, H., Thompson, L. M., Waldeck, N., Gross, D. S., and Mooney, D.
Conformational changes in rat liver chromatin after liver regeneration. Biochem.
J., 793:671-678,
1981.
26. Sinha, B. K., and Chignell, C. F. Binding mode of chemically activated semiquinone-free radicals from quinone antica.icer agents to DNA. Chem.-Biol.
Interact., 28:301-308,
1979.
27. Solle, T. N., and Yuncker, C. Adriamycin induced changes in translocation of
sodium ions in transporting epithelial cells. Life Sci., 22:1907-1920,1978.
28. Thompson, L. M., Arquilla, M., and Simpkins, H. The interaction of platinum
618
29.
30.
31.
32.
33.
34.
35.
36.
37.
complexes with nucleosomes investigated with fluorescent probes. Biochim.
Biophys. Acta, 698: 173-182,1982.
Topal, M. D., and Fresco, J. R. Fluorescence of terbium ion-nucleic acid
complexes: a sensitive specific probe for unpaired residues in nucleic acids.
Biochemistry, Õ9:5537-5542,1980.
Tritton, T. R., Murphree, S. A., and Sartorelli, A. C. Characterization of drugmembrane interactions using the liposome system. Biochem. Pharmacol., 26:
2319-2323,1977.
Tritton, T. R., Murphree, S. A., and Sartorelli, A. C. Adriamycin: a proposal on
the specificity of drug action. Biochem. Biophys. Res. Commun., 84:802-808,
1978.
Tritton, T. R., and Yee, A. The anticancer agent Adriamycin can be actively
cytotoxic without entering cells. Science (Wash. D. C.) 277: 248-250, 1982.
Waldes, H., and Center, M. S. The interaction of Adriamycin with nuclear DNA:
evidence for a drug induced compaction of nuclear chromatin. Biochem.
Biophys. Res. Commun., 98: 95-101,1981.
Weltman, J. K., Szaro, R. P., Frackelton, A. R., Jr., Dowben, R. M., Bunting,
J. R., and Cathou, R. E. N-<3-Pyrene)maleimide: a long lifetime fluorescent
sulfhydryl reagent. J. Biol. Chem., 240: 3173-3177,1973.
Zimmerman, S. B., Cohen, G. H., and Davies, D. R. X-ray fiber diffraction and
model-building study of polyguanylic acid and polysinosinic acid. J. Mol. Biol.,
92: 181-192,1975.
Zwelling, L. A. Michaels, S., Erickson, L. C., Unserieider, R. S., Nichols, M.,
and Kohn, K. W. Protein-associated deoxyribonucleic acid strand breaks
in U 210 cells treated with deoxyribonucleic acid intercalating agents 4-{9acridinylamino)methane sulfon-m-c/s-anisidide and Adriamycin. Biochemistry,
20:6553-6563,1981.
Zunino, F., DiMarco. A., Zaceara, A., and Gambetta, R. A. The interaction of
daunorubicin and doxorubicin with DNA and chromatin. Biochim. Biophys.
Acta, 607. 206-214,1980.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.
VOL.
44
Effects of Adriamycin on Supercoiled DNA and Calf Thymus
Nucleosomes Studied with Fluorescent Probes
Henry Simpkins, Leslie F. Pearlman and Leslie M. Thompson
Cancer Res 1984;44:613-618.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/44/2/613
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1984 American Association for Cancer Research.