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Introduction
Daunomycin is an anthracycline antibiotic widely
used in the treatment of myelogenous leukemia and solid
tumors[1,2]. Numerous studies have revealed that nuclear DNA is an important target for this drug. The structure of
daunomycin consists of 2 distinct domains (Figure 1): a planar aglycon chromophore that intercalates between adjacent base
pairs of DNA and an amino sugar ring that lies in the minor groove of the DNA double
helix[3,4]. Binding of daunomycin to DNA results in the inhibition of both, DNA replication and RNA
transcription[5_7].
In the cell nucleus, DNA is compacted into chromatin, which is a complex structure built from repeating units
(nucleosomes)[8]. These consist of 145 bp DNA wrapped around an octamer of basic proteins (core histones). The core histones are small,
basic proteins ranging between 11 and 16 kDa, with more that 20% of their amino acids composition being lysine and
argenines. There are 5 main histones: the linker histones of the H1 family and the 4 core or nucleosomal histones H2A, H2B,
H3, and H4, which associate as H2A/H2B dimers and a
H3/H4 tetramer, arranged in a octamer form: the DNA is wrapped
within the nucleosome-chromatin unit[9].
How the occurrence of histones that bind to DNA
affects the binding of daunomycin, is an important question
when trying to understand the mechanisms of action of this
drug at the chromatin level. To explore this question, some
authors have focused on the binding of daunomycin to
chromatin and nucleosomes and have shown that the binding
affinity of daunomycin to chromatin increases upon the
removal of histones[10_12]. We recently focused on the
interaction of daunomycon with the histone H1 family (linker
histones) in chromatin and in solution, providing evidence
that the binding is cooperative and stabilizes the protein
against thermal denaturation[13_15].
In the present study, we have extended our research on
the interaction of daunomycin with the core histones free in
solution and cross-linking with a bifunctional reagent to
produce a complex designated, cross linked core (CL-core)
resembling an octamer in nucleosomes. The results provide
an evidence that daunomycin binds to free core histones to
a greater extent than to the CL-core complex, implying that
the environment of core histones in the chromatin plays a
fundamental role in the drug-histone interaction.
Materials and methods
Chemicals Daunomycin hydrochloride (Figure 1A) was
purchased from Sigma (St Louis, MO, USA). A stock
solution of drug was prepared in sterile distilled water at a
concentration of 2 mg/mL and stored at -20 ºC until further use.
A dilution of the drug stock in the appropriate buffer was
prepared immediately before use. The concentration of
daunomycin was determined spectrophotometrically using
an extinction coefficient of 11500
mol-1· cm-1 at 480 nm.
Preparation of histones The total histone containing all
5 histones was extracted from calf thymus by 0.25 mol/L HCl
described by Johns[16]; the core histones were further
purified using CM-Sephadex-C25 ion exchange chromatography
(Pharmacia)[17]. The purified core histone was dissolved in
20 mmol/L Tris buffer (pH 7.3) and after pH adjustment; the
solution was stored at -20 ºC and used within a month.
Extensive dimethyl suberimidate (DMS: Sigma, St Louis,
MO, USA) cross-linked with the thymus core histone octamer
was formed by cross-linking of histones with DMS in 2 mL
NaCl and 0.1 mL sodium borate (pH
9)[18]. The cross-linked octamer was purified further by preparative gel
electrophoresis and the electroelution technique. After electrophoresis,
the protein complex was electroeluted for 8 h at 80_100 V at
4 ºC using dialysis tubing (3500 cut-off). The sample was
then centrifuged for 15 min at 3000×g and the clear
supernatant dialyzed extensively with 20 mmol/L Tris-HCl (pH 7.3)
for 96 h by changing the dialysate every 6 h. After brief
centrifugation, the supernatant was concentrated by a
VIVASPIN filter (5000 cut-off, Vivascience, Lincoln, UK),
dialyzed against 20 mmol/L Tris buffer and stored at -20 ºC
until use.
The purity of the proteins was checked on 15 %
SDS-polyacrylamide gel electrophoresis as described by Lammeli
(Figure 1B)[19]. The protein concentration was determined
according to Bradford[20] using bovine serum albumin as a
standard.
Interaction of daunomycin with histones To obtain the
correct time of incubation, the proteins were incubated with
various concentrations of daunomycin and after different
time intervals, aliquots were taken and the absorbancies were
monitored at 210 and 480 nm.
The purified core histone and CL-core were dissolved in
20 mmol/L Tris-HCl buffer (pH 7.3) and their concentration
was determined spectrophotometrically. An appropriate
concentration of daunomycin was incubated with the proteins
(3_150 µmol/L) for 30_60 min at 23 ºC in the dark. Free
daunomycin and histones were prepared in the same buffer and
incubated along with the drug-histone samples under the
same conditions and used as a control.
UV/Vis spectroscopy Core histone (or CL-core) and
daunomycin were mixed as described above in 20 mmol/L
Tris buffer (pH 7.3), and the spectrophotometric
measurements were carried out at 23 ºC. The absorbancies were
measured at 210, 230 and 480 nm using a UV-260 Shimadzu
spectrophotometer (SHIMADZU Corporation, Kyoto, Japan)
and the results were normalized for the protein concentration.
Spectra were recorded between 190 and 250 nm.
Fluorescence spectroscopy The measurements were
performed on a fluorescence spectrophotometer (Hitachi
MPF-4, Japan) equipped with a thermostatically controlled
cell holder at an ambient temperature. The monochromatic
slits were set at 5 nm to reduce the intensity of the signal
depending on the experiment. All samples were made in 20
mmol/L Tris-HCl (pH 7.3) at 20 ºC and a quartz fluorescence
cell with a 1 cm path length was used. Protein solutions
(5_10 µmol/L) were titrated with aliquots of daunomycin (0_50
µmol/L) and equilibrated until a steady emission reading was
obtained. The accumulated volume of titration was less than
10 µL, so the dilution effect was negligible.
Daunomycin and the histones were prepared
individually in the same buffer and used as a control. Amino acid
tyrosine was also dissolved in the same buffer and its
emission spectrum was recorded in the same condition and used
as a control. The spectra were recorded between 290 and
370 nm after the excitation at 278 nm. The
(Io_I /Io) values for
each sample were normalized with respect to the
fluorescence of the protein in the absence of the drug in which
Io and I are fluorescence intensity before and after the addition
of daunomycin, respectively.
Equilibrium dialysis The proteins in 20 mmol/L Tris-HCl
buffer (pH 7.3) were dialyzed against the buffer containing
serial concentrations of daunomycin using Scientific
Instrument Center dialysis tubing at room temperature. The
equilibrium was achieved within 72 h with the dialysis tubing.
The total drug concentration (Ct) and the concentration of
the free drug (Cf) in the dialysate were measured directly
from the absorbance at 480 nm before and after dialysis
using the extinction coefficient of 11500
mol-1· cm-1. The amount
of the bound drug (Cb) was obtained from
Cb=Ct-C
f. Binding parameters were determined from the plot of
r/Cf versus r according to the Scatchard
method[21] where r is the ratio of
the bound drug to the molar concentration of
proteins. The Scatchard plot gives an
x-intercept of n, where n is the
apparent number of binding sites and K (apparent binding
constant) corresponds to the negative value of the slope of
the curve. Also using this equation;
1/r=1/n+Kd/nCf
,
r versus Cf was drawn. The Hill coefficient
(nH) was determined from the slope of the ln
(Cf) versus ln (r/n_r)
according to the Hill equation[22].
Results
In the present work, the binding of the anticancer drug,
daunomycin, to core histones in 2 different states, core
histones free in solution and core histones cross linked with
DMS (CL-core), has been investigated and compared for the
first time. As seen in Figure 1B, core histones represent 4
bands corresponding to histones H3, H2A, H2B and H4 and
the purified protein lacks histone H1. The cross linked
histone designated as CL-core, shows a histone complex with a
molecular weight of about 105 kDa which is similar to the
octamer size of nucleosomes. Also, the time study
experiment presented in Figure 2 shows that the interaction of
drug with the core histone is completed between 30 and 60
min. Therefore this incubation time range was used
throughout the experiments.
Comparison of the fluorescence profiles of daunomycin
interaction with core histones and CL-core The
fluorescent emission spectra obtained from the interaction of
daunomycin with the core and CL-core is shown in Figure 3. Figure
3A shows the fluorescence emission spectra of the core
histone in the presence and absence of various concentrations
of daunomycin. The fluorescence spectrum of tyrosine has
also been provided for comparison. As was expected, core
histones free in solution, exhibit emission spectra in the
position corresponding to tyrosine with a maximum intensity at
305 nm. The addition of daunomycin to the histone solution
reduces the fluorescence intensity of the protein without
any red shift in the emission maxima
(Imax) as the drug concentration is increased.
Figure 3B represents the fluorescence emission spectra
of CL-core in the absence and presence of daunomycin. It is
shown that the fluorescence intensity of the protein is also
decreased as the drug concentration is increased, but the
extent of reduction is less than that of core histones free in
solution. Furthermore, for comparison, the effect of
daunomycin on the fluorescence emission intensity of the
individual histone pairs, H2A_H2B and H3_H4 was also
investigated in the same condition. The spectra are given in
Figure 3C. When the fluorescence intensities are normalized
to protein concentration and
Io_I/Io is
calculated, the core histones still exhibit the highest emission intensity
reduction and the order of intensity reduction is: core
histones>H3_H4>CL-core, (Figure 3D). This clearly implies that the
binding of daunomycin to core histones is dependent on
their accessibility to the environment.
UV/Vis spectroscopy analysis Absorbance in the
UV/Vis region has been successfully used for the analysis of
daunomycin_DNA or linker histone[4,16] interactions. In this
study, the purified proteins were incubated in the presence
and absence of various concentrations of daunomycin in
the dark; the changes in their absorbance at 480 and 210 nm
were measured, and the data were normalized with respect to
the protein concentration. Figure 4A shows the absorbance
changes of the core histones as a function of the drug
concentration. As can be seen, the absorbance of the core
histones at 210_220 nm considerably decreased upon the
addition of the drug (Figure 4A). This absorbance reduction
is also observed in the pattern of 480 nm, which is
exclusively related to the wavelength of daunomycin. Difference
spectra patterns also confirm the above results and indicate
that upon the addition of daunomycin to the protein
solutions, hypochromicity occurs, while CL-core (Figure 4B)
exhibits much lower hypochromicity in the presence of the
drug than the core histones free in solution (Figure 4C). The
results indicate that daunomycin inters the sites of histones,
which are active in electron excitation.
Cooperative binding of daunomycin to core
histones The binding isotherms obtained from the equilibrium dialysis is
shown in Figure 5. In both cases, the Scatchard plot exhibits
a cooperative binding behavior, as illustrated by the
positive slope observed in the low r regions of the binding
isotherms. The curve reaches a maximum at a value
of r =0.8 (core histones free in solution) and
r=1.3 (CL-core). A decrease in the slope is observed at higher
r values. Although core and CL-core exhibit similar but not identical binding
isotherms, the later shows lower r/C
f values, which is indicative of a much lower binding affinity of daunomycin to
CL-core. Indeed, the binding constant (K) for the CL-core
(K=5.2×105 mol-1)
is relatively lower than that obtained for the
core histones (K=6.5×105
mol-1). Drawing ln r/n-r
against ln Cf gives a straight line with a slope of the
nH (Hill coefficient). The
nH values of 2.25 and 1.79 were calculated for the core
histone and CL-core, respectively, which demonstrate that
daunomycin has lower binding affinity to the compact
structure of CL-core compared to the open structure of core
histones free in solution.
As shown the Figure 5B, a plot of r against
Cf is sigmoid; that is, as
Cf increases, r rises slowly at first, then rapidly and
finally levels off, indicating that the system approaches to
equilibrium or saturating state. Using the equation of
DGo= -RT ln K, total macroscopic free energy values of
DGo=-33 kcal/mol and DGo=-23.6 kcal/mol were obtained for the
core histones and CL-core respectively. The occurrence of
a negative Gibbs free energy suggests that the interaction
process is exergonic.
Binding isotherms also confirm the above results.
Although both samples, core histones and CL-core, show
positive cooperativity, but daunomycin still shows lower
binding affinity to CL-core. This is obvious when the
r/Cf , K and
DGo values are compared.
Discussion
The anticancer activity or cytotoxic effects of
anthra-cycline antibiotics may involve the interaction of these drugs
with the nuclear components. The binding of daunomycin
to DNA and nucleosomes has been studied in
detail[3_8,12]; whereas the binding of antitumor drugs to the protein
components of chromatin is still questionable, and only a few
reports has been published besides those dealing with the
interaction of these drugs with
topoisomerases[23]. During the past few years our laboratory has focused on the
interaction of antitumor drug, daunomycin, with the histone H1
family (linker histones) in
solution[13,14] and in
chromatin[15]. The results come in support of the notion that the protein
component of chromatin can also be considered as a target
for the activity of these antitumor drugs. Moreover, this is
the first paper demonstrating the binding behavior of
anticancer drug, daunomycin, to core histones in the absence of
DNA.
Structural studies have shown that core histones are
comprised of 2 distinct domains. The histone fold domain
(hydrophobic core), formed by 3 α-helices connected by 2
loops is involved in histone_histone and histone-DNA
interactions, while the N-terminal tail domain is composed of
about 15_30 highly basic amino acids, which expels out of
the nucleosome surface[24,25]. Under physiological
condi-tions, core histones interact with each other to form a
heterotypic octamer consisting of a histone H3_H4 tetramer
and two histone H2A_H2B dimers, which constitute the
protein core of the basic chromatin subunit, the nuclear core
particle. In solution and in the absence of DNA, the histone
octamer exists in equilibrium between its constitutive
H2A_H2B dimers and the H3_H4 tetramers, which have been
extensively characterized[25, 26].
As mentioned earlier, the fluorescence emission
intensity of all proteins used here, in the absence of daunomycin
exhibit a characteristic fluorescence emission intensity
maximum at 305 nm corresponding to the maximum fluorescence
emission of tyrosine[27]. In contrast to histone H1, which has
only 1 tyrosine[28], core histones contain a higher content of
tyrosine. In this case, histones H3 and H4 have 2 tyrosine
and H2A and H2B contain 3_4 tyrosines (sequence analysis
using Swiss Prot or Expasy programs). This aromatic amino
acid is mostly located in the hydrophobic or globular part of
the histones, whereas, tyrosine 38 and 122 of H2B and 39 of
H2A are exposed to the solvent. Therefore the reduction in
the emission intensities, in the presence of the drug, is as a
result of fluorescence quenching. Most likely, in CL-core,
the tyrosines are hidden inside the globular part of the
protein (as a result of cross linking) providing less fluorescence
quenching. Despite the ability of H2A_H2B and H3_H4 to
interact with daunomycin in solution, neither of them
appears to be accessible to the drug in the nucleosome
environment[15]. This is confirmed by the lower affinity of
daunomycin to CL-core.
Results of absorption spectroscopy have shown that
daunomycin binds to both core histones and CL-core in
solution and reduces the absorbancies at 210 and 480 nm
(hypochromicity). Binding isotherms also confirm the above
results. Although both samples, core histones and CL-core,
show positive cooperativity, daunomycin still shows lower
binding affinity to CL-core. This is obvious when the
r/Cf , K and
DGo values are compared. Although our results clearly
demonstrate the interaction of daunomycin with the core
histones in solution, but the mode of their interaction still
remains obscure. From the results presented, both
hydrophobic and electrostatic interactions can be speculated.
Summing up, the present study clearly demonstrates the
lower affinity of daunomycin to CL-core rather than to free
core histone. The result is in agreement with what we have
obtained for the chromatin[12,15,29], in which the core histones
covered with DNA are not accessible to the drug. We
propose that, within the chromatin context, it appears that
binding of daunomycin to core histones is dependent on their
accessibility to the environment. This is particularly
significant in the case of active chromatin and tumor cells.
Neoplastic cells have a high transcription activity so that most
of their chromatin exists as euchromatin. Such alteration in
cancerous cells enhances the accessibility of the drug to the
protein components of chromatin (core histones). This also
coincides with our previous results showing that adriamycin,
a similar drug to daunomycin, has higher affinity to active
chromatin compared to inactive
chromatin[29]. Consequently, the binding of daunomycin to DNA on one hand and to core
histones on the other, produces a compact structure that
inhibits chromatin metabolism, such as transcription and
replication. Another explanation is that in active chromatin
and in tumor cells that histones are highly acetylated,
promoting the accessibility of the histones to the environment,
it can be speculated that anthracycline antibiotics
preferentially bind to acetylated histones, thus the complex prevents
histone deacetylation. As a result, certain genes including
apoptotic_related genes are continually expressed and the
cells proceed into apoptosis. Although comparison of the
interaction of daunomycin with acetylated and unacetylated
histones, can provide important information about the real
action of daunomycin at the chromatin level, extensive work
needs to be elucidated.
Acknowledgements
The authors would like to thank Marzeyeh
YOUSOF-MASBOOGH for her excellent research assistance.
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