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The anthracycline antibiotics:antitumor drugs that alterchromatin structureAzra Rabbani,1 Ron M. Finn,2 and Juan Ausio2*

SummaryAnthracycline antibiotics are an important group ofantitumor drugs widely used in cancer chemotherapy.However, despite the increasing interest in these che-motherapeutic agents, their mechanism of action is notyet completely understood. Here, we review what iscurrently known about the molecular mechanisms in-volved with special emphasis on the interaction of thesedrugs with chromatin and its constitutive components:DNA and histones. The evidence suggests that one veryimportant component of the activity of these drugs is

the result of these manifold interactions that lead to achromatin unfolding and aggregation. This chromatinstructural disruption is likely to interfere with the meta-bolic processes of DNA (replication and transcription)and it may play an important role in the apoptosis under-gone by the cells upon treatment with these drugs.BioEssays27:50 56, 2005. 2004 Wiley Periodicals, Inc.

Introduction

Although anthracycline antibiotics were recognized for their

antibacterial properties in 1939, their chemical characteri-

zation experienced a substantial increase only after the

therapeutic value of their antitumor activity was described in

the early 1960s. Two of these compounds daunomycin

(daunorubicin) and adriamycin (hydroxyl daunomycin or

doxorubicin) were initially isolated fromStreptomyces peuce-

tius varcaesitue(1,2) and exhibit the widest spectrum of

antitumor activity against human cancers.(3,4) Because of the

increasing potential medical relevance of these drugs in cancer

chemotherapy, there has been a lot of interest in chemically

modifying them to try to minimize their cardiotoxic side

effects.(4,5) In this regard, many anthracycline derivatives

have been produced in recent years.(6,7) A good example can

be found withpixantrone {6,9-bis[(2-aminoethyl) amino] benzo

(g) isoquinoline-5, 10-dione dimaleate}, which has already

gone through phase II clinical trials and which shows a lot of

promise for the treatment of Non-Hodgkins lymphoma.(8)

Daunomycin andadriamycinwitha chemicalformulaof C27H29 NO10 andC27 H29 NO11, respectively, are soluble in water,

ethanol and methanol. They are planar molecules consisting

of a tetracycline ring bound to a daunosamine through a

glycoside bond. The quinone and hydroquinone groups ofthese molecules act as electron donors and acceptors.(9)

Anthracyclineantibiotics and their derivatives are an integral

component of the current chemotherapy in the treatment of

solid tumors and leukemia; indeed daunomycin has beenused

primarily to treat adult myelogenous leukemia.(10,11) Although

there is still some controversy about their mode of action,

the anticancer activity or cytotoxic effects may involve the

interaction with nuclear components, especially DNA and type

II topoisomerases;they mayalso participate in theinhibition of

DNA and RNA synthesis.(9,1214)

Characterization of the binding sites of antitumor drugs in

the cell is important because it can provide clues required for

further drug design to enhance drug efficacy. This reviewfocuses its attention on progress made on the understanding

of the interaction of anthracycline antibiotics adriamycin and

daunomycin with different chromatin components.

Interaction with DNA: Binding of sequence

specific intercalators

There is compelling evidence that cellular DNA is the primary

target for these drugs. Quantitative microspectrofluorometry

has been used to show that adriamycin rapidly accumulates

in the nuclei of living cells and is nearly completely bound to

DNA.(15) Flow cytometry has shown that daunomycin is

intercalated into DNA within the cell.(16) Furthermore, the

surface-enhanced Raman spectrum of adriamycin within

the nuclei of erythroleukemia cancer cells was found to

resemble that observed in vitro forthe drug DNA complex.(17)

Together with many other reports (see below) all this clearly

suggests that anthracycline antitumor antibiotics are strongly

associated with nuclear DNA in vivo. From a mechanistic

point of view, drugs that interact with DNA can be divided into

50 BioEssays 27.1 BioEssays 27:5056, 2004 Wiley Periodicals, Inc.

1Institute of Biochemistry and Biophysics, University of Tehran, Iran.2Department of Biochemistry and Microbiology, University of Victoria,

Canada.

Funding agency: This work was supported by a grant of the Research

Council of the University of Tehran to A.R. and by a Canadian

Institutes of Health Research (CIHR) grant to J.A. (MOP-57718).

*Correspondence to: Juan Ausio, Department of Biochemistry and

Microbiology, University of Victoria, PO Box 3055, Petch Building

Room 220, Victoria, BC, V8W 3P6, Canada. E-mail: jausio@uvic.ca

DOI 10.1002/bies.20160

Published online in Wiley InterScience (www.interscience.wiley.com).

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two broad categories: intercalating drugs and those that

chemically bind to DNA. Anthracycline antibiotics are classi-

fied in the first group. Intercalators bind to DNA by inserting

their planar chromophores between DNAbases. In addition to

stacking and hydrophobic interactions, the intercalation can

be further stabilized by hydrogen bond formation in-between

the bases of DNA, a situation where the sugar moieties are

interacting withthe central chromophores of the drug.(9,18)The

anthraquinone ring intercalates between DNA base pairs, with

itslongaxis nearly perpendicular to theaxisof thedoublehelix.

One of the rings acts as an anchor and stabilizes the complex

through hydrogen bond interactions as the daunosamine

sugar lies in the minor groove.(18) The occurrence of a single

positive charge on daunomycin contributes electrostatically to

the binding.(9)

Binding of daunomycin and adriamycin to DNA has been

extensively characterized.(1921) Equilibrium dialysis, ab-

sorption and fluorescence spectroscopy and phase partition

methods have shown the occurrence of a negative coopera-

tive binding isotherm pattern in the formation of the daunomy-cinDNA (or adriamycinDNA) complex.(19,20) However, our

results and those of others using the same technique have

demonstrated the occurrence of positive cooperative binding

isotherms.(2224) These controversial results could be due to

differences in the DNA sequence and/or experimental condi-

tions used. The magnitude of the binding constant of

daunomycin to DNA is strongly dependent on the base pair

composition of DNA. Both drugs exhibit a strong structural

specificity in their interaction with DNA. Using equilibrium

dialysis and DNaseI footprinting techniques Chaires et al.(25,26)

haveshown thatthe preferredsite of intercalationmust contain

adjacent GC base pairs and the optimal daunomycin-binding

site may span over a triplet sequence, constituting of anAT base pair at the 50-position, flanked by two contiguous GC

base pairs. Physically the drug covers three base pairs

preferentially 50-A/TCG.(25,26) The preference is due to the

specificity of the hydrogen-binding interactions. The C-9

hydroxyl group of daunomycin forms a particularly strong

double hydrogen bond with N-2 and N-3 of the guanine at the

center of the preferred triplet-binding site.(27) The sequence

specificity of the daunomycin DNA interaction(26) also reveals

that the affinity of daunomycin toward natural DNA increases

with its GC content. The specificity of daunomycin DNA

interaction has also been determined by high-resolution

transcription assays.(9,28,29)

The kinetic analysis of the formation of the daunomycin

DNA complex shows that this is a slow and complex

process.(30,31) The average lifetime of the-binding is approxi-

mately one second and several steps are required to describe

the time course of its DNA-binding reaction in contrast to what

is observed with other intercalators. This complicated binding

kinetics exhibited by daunomycin is most likely the result of its

preferential binding to certain DNA sequences.

Chen et al.(32) have performed computational analysis to

model the interaction of daunomycin with six duplex hexanu-

cleotides of defined sequence. They inferred from their calcu-

lations that the most energetically favorable binding sites for

these antibiotics were the triplet sequences of 50-TCG and 50-

ACG.(25,32) However, Chaires et al.(25) have shown that the

requirement for A or T at the 50 end is not absolute and that

three contiguous GC base pairs form an acceptable binding

site although possibly with weaker binding affinity.

The interaction of daunomycin with left-handed Z-DNA

has also been the subject of many studies(3335) that have

confirmeda strong preference forthe B-form of DNA.Allosteric

conversion from Z-DNA to a right-handed conformation by

daunomycin has also been reported.(35) Binding of daunomy-

cin to poly (dGdC) under ionic strength conditions that would

favor the left-handed Z conformation of this DNA, effectively

inhibits the B-to-Z transition and reverts Z-DNA conformation

to theright-handedform.Conversion ofDNA toa form different

from the standard right-handed B conformation results in a

dramatic reduced affinity for daunomycin.(35)

Binding of daunomycin and adriamycin to DNA increases

Tm of DNA melting point by 308C as saturation of the potential

binding sites is reached.(19) Increase in Tm values of DNA is

due to stabilization of DNA double helix resulting from the

intercalation of the drug and can be used to explain DNA and

RNA synthesis inhibition, as well as the effects of these drugs.

Interaction with the chromatin:

unfolding transitions that lead to aggregation

It is obvious thata simple intercalationmodel for the interaction

of the drugs with DNA is not enough to explain their overall

mechanism of action in the cell. In the cell nucleus, DNA does

notexistas a nakedstructure butis associated with a variety ofproteins making a nucleoprotein complex called chromatin.(36)

The mechanism of interaction and possible intracellular

targets of antitumor drugs are often inferred from studies of

the affinity of these drugs for free DNA in solution. Yet the

tertiary structure and environment of DNA in the cell can

significantly modulate these interactions.

Equilibrium dialysis, hydrodynamic and electric dichroism

techniques have been used to investigate the effects of

anthracycline antibiotics on chromatin. Binding isotherms

obtained from the interaction of daunomycin with 175 bp

nucleosomes (depleted of histone H1) at several ionic strength

conditions suggest a negative cooperative pattern and a

strongly reduced binding relative to free DNA.(3740) It was

shown that the affinity of daunomycin for different nucleohis-

tone complexes is in the following order: DNA>175bp H1/H5

depleted nucleosomes>nucleosome core particles.(14,38)

These results are in agreement with those observed for the

interaction of daunomycin with chromatin containing either

the full native linker histones (H1 and H5) or depleted of these

histones.(21) Addition of daunomycin to 175 bp H1-depleted

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nucleosomes or 146 bp nucleosome core particles stabilizes

their low melting transition temperature, which corresponds to

the regions of the nucleosomal DNA less tightly bound to

the histone octamer at the flanking ends of the particle. (38)

Comparative studies between H1-depleted 175 bp nucleo-

somes and their constitutive DNA using circular dichroism(40)

and hydrodynamic studies(38) have provided additional useful

structural information. Binding of anthracyclines increases the

ellipticity of thepositive band at 275nm in nucleosomesand to

lesser extent in DNA. While with DNA this increase can be

attributed to changes in the winding angle of the double helix

resulting from the intercalation of the drug,(41) the relatively

larger change observed with nucleosomes is most likely the

result of DNA additionally becoming freed of the histone

interaction constraints.(42) Consistent with the changes in

thermal stability of the nucleosome just described, the region

of DNA affected should correspond to the entry and exit sites

into the nucleosome becoming stiffer as a result of the

intercalation of the anthracycline antibiotic. Such release of the

DNA flanking ends would result in an increase of the frictionalparameters of the nucleosome as it has been experimentally

shown by the decrease observed in the sedimentation

coefficient that accompanies the initial titration of the nucleo-

somes with the drug. Titration beyond 0.15 molecules of drug/

bp of DNAresults in a reversal of thesedimentation coefficient

trends, in fact, it starts to increase beyond this point as a result

of nucleosome aggregation.(38) The hydrodynamic results

obtained with native or linker histone-depleted chromatin

fibers(21) mirror these results and provide a further insight into

the molecular mechanisms involved in the interaction of

anthracyclines with chromatin (see Fig. 1).

The hydrodynamic behavior of chromatin complexes

has been extensively characterized.(43) The ionic-strength-dependent folding of the polynucleosome fiber is highly

dominated by the occurrence of linker histones (histone

H1)(44) and only a limited extent of folding is reached in its

absence.(43) Upon addition of daunomycin to nativeor to linker

histone-depleted chromatin complexes, a biphasic transition

in the sedimentation coefficient is observed in both in-

stances,(21) similar to what is observed for the individual

nucleosomes.(38) The inflection point of the transition de-

creases with the ionic strength and with the presence of linker

histones. This reflects the decrease of free linker DNA

available for the interaction with the drug under these con-

ditions and the preference of anthracycline drugs for free

versus nucleosomal DNA.(38) Our interpretation of these

results is shown in the model proposed in Fig. 1B and 1C.

Intercalation of the drug takes place at the free DNA in the

linker DNA regions, changing the linker twist(45) and gradually

extending toward the histone octamer, hence, resulting in an

unfolded chromatin conformation. Binding of the drug to the

nucleosomal DNA is impeded by the core histone tails.(46)

Once a critical amount of DNA has been unraveled, the

nucleosome subunits startlosing their stabilityand, as a result,

the nucleoprotein complexes start aggregating in a process

that is reminiscent of the nucleosome aggregation observed

in vitro when nucleosomes are trimmed with micrococcal

nuclease beyond 140 bp.

The chromatin unfolding transitions and final aggregation

appears to occur without any noticeable dissociation of

histones(21) and the conformation of the histone octamer is

little affected by the whole process as reflected by the lack of

spectral changes in the far UV region of the CD spectrum. (41)

Our results also indicate that daunomycin has a destabiliz-

ing rather than an inhibitory effect on chromatin folding(21) and

easily explain the unwinding/aggregation process described

by Cera and Palumbo.(47)

Binding of anthracyclines to

chromosomal proteins

A questionstillto be consideredis: do antitumor drugs alsobind

to any of theprotein components of chromatin? Unfortunately,

only a few limited reports have been published besides thosedealing with the interaction of these drugs with topoisome-

rases.(48) Some recent studies have focused their attentionon

the interaction of antitumor drugs adriamycin and daunomycin

with histone H1 (linker histones).

Using a combination of absorption and fluorescence

spectroscopy, thermal denaturation and equilibrium dialysis

techniques,(49,50) it has been shown that similar to what is

observed for the interactions with DNA,(19) the binding of

daunomycin to histone H1 stabilizes the protein against

thermal denaturation.(49) This is in contrast to the binding of

adriamycin to HMGB1, which destabilizes the protein against

thermal melting(A Rabbani,RM Finn andJ Ausio, unpublished

data). The binding isotherms for H1-daunomycin complexalways exhibit a positive cooperativity and point to the

existence of two binding sites. The occurrence of a negative

Gibbs free energy suggests that the interaction process is

spontaneous (exergonic).(50) These results come in support

of the notion that the protein components of chromatin (i.e.

histone H1) can also be a target for the activity of these

antitumor drugs. Indeed, recent results from our own labo-

ratory have shown that daunomycin is able to specifically

interact withchromatin-bound histone H1(ARabbani,RM Finn

and J Ausio, unpublished data). Apart from histone H1, the

interaction of anthracycline antibiotics withtopoisomerases(48)

and nonhistone proteins have also been reported.(5153)

Binding of daunomycin to nuclear nonhistone proteins

within regulatory regions of the genome (for instance in the

case of topoisomerases) can interfere with the synthesis of

macromolecules essential for cell proliferation.(54) Therefore,

daunomycin may exert a dual effect on cell proliferation both

regulating and modifying the template activity of DNA that

would be required for the normal RNA synthesis and DNA

repair. Mechanistically, such drugs may block the activity of

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processive enzyme complexes and/or interfere withthe access

of transactivating factors to DNA.(55,56) Indeed anthracyclines

have been shown to inhibit theactivity of topoisomerasesI and

II(48,57) impairing the DNA repair and DNA replication activities

of the cell.

Concerted drug-induced structural changes of

chromatin leading to apoptosis

Althoughmost of theinteraction of the anthracyclineantibiotics

involves intercalation between the bases in DNA, these drugs

can also alter the structure and function of this molecule (and

hence chromatin) in many different ways. These structural

changes may ultimately contribute to the apoptotic process

induced by these drugs in cancer cells(58,59) (see Fig. 2).

One such change involves the covalent modification of

DNA by daunomycin.(60) The formation of anthracyclineDNA

adducts hasbeenobservedto occur both in vitro andin vivo(60)

through a mechanism that involves the iron-complex of the

drug.(61) The formaldehyde production resulting from the

oxidative stress induced by the drug metal complex(61,62)

produces a covalent attachment to theG-basesof DNA(61) that

functions as a virtual interstrand crosslinker.(62) Such DNA

crosslinking could participate in the chromatin aggregation

process occurring during apoptosis.

Figure 1. A: Hydrodynamic analysis of the changes in chromatin conformation. The sedimentation coefficient (s20,w) of chromatin (*),

linker histone-depleted chromatin (*) and corresponding DNA (~) are plotted as a function of the daunomycin:bp ratio (r). The analysis

shown was performed at75 mMNaCl in10 mMTrisHCl (pH 7.5), 0.1 mMEDTA buffer. B: As daunomycin binds (intercalates to DNA) in

thelinkerregionsof chromatin,it alters thelinkertwist (redarrows)(45) changingthe orientationof adjacentnucleosomes. C: (I) Chromatin is

in a folded nativelike conformationunder the experimental ionicconditions usedin A.(43) Thisstructure correlates withthe portionsof the

plots illustratedin A) andlabeledas 1. (II) The decreaseobserved in the sedimentation coefficient of chromatin as r increases, is indicative

of a chromatin unfolding. This unfolded chromatin correlates with the portions of the plots illustrated in A) that are labeled as 2. This is the

result fromthe daunomycin(shownin red)intercalationin thelinkerDNA (seeFig. 1B).(III) Furtherincrease in r resultsin a sudden increase

in s20,w[see the portions of the plots illustrated in A) that are labeled 3] which is due to aggregation of the chromatin complexes.

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The chelation of ions by anthracyclines can also result in

the generation of reactive oxygen species (ROS), which is

responsible for the free radical cytotoxicity of these drugs.(63)

As it occurs with other quinines, these antibiotics can be

enzymatically reduced to semiquinone radicals, which, in the

presence of oxygen and iron, can result in the production of

highly DNA damaging OH-radicals.(63)

The intercalation process itself results in a distortion of

the DNA conformation that causes the inhibition of Topo II.(64)

The effects of anthracyclines on this enzyme(65) have been

extensively studied (see(66) for a review). In this instance, the

mechanism of action involves the binding of the drug to the

Topo IIaDNA complexes forminga ternary complex.(67)Such

binding interferes with the re-ligation step of the topoisome-

rase resulting in double-stranded DNA breaks.(66)

Concluding remarks

As has been described in the previous sections, DNA

intercalation of anthracyclines leads to changes in chromatin

supercoiling that eventually result in chromatin aggrega-

tion.(23) DNA intercalation still remains one of the main

targeting sites for the binding of these drugs to DNA.

Therefore, in closing, we would like to propose that such

aggregation, in addition to the chromatin structural changes

described above, may contribute in a concerted way to the

apoptotic cell death induced by these drugs.(58,59) Chromatin

aggregation(23) may facilitate or promote the DNA fragmenta-

tion(68,69) by specific apoptotic endonucleases. Indeed, it has

been recently shown that chromatin aggregation precedes

oligonucleosomal fractionation(70) andthatDNA is supercoiled

in apoptotic chromatin.(71)

Despite all this, the reason(s) why or how anthracyclines

exerttheir preferential killing of tumor versus non-carcinogenic

cells are still not well understood. It is likely that the manifold

effects described above not only mediate the action of these

drugs in killing cancer cells but are also responsible for their

inherent side effects e (i.e. cardiotoxicity).(4,5) A line of thought

is that their selectivity for the cancer cell targeting resides in

Figure 2. Anthracycline DNA interaction results in manifold alterations of chromatin structure. The concerted action of these changes

are most likely directly or indirectly responsible for the chromatin aggregation that precedes the chromatin fragmentation that is

characteristic of apoptotic cells. The red arrow simply points to intercalation being one of the major components of the anthracycline drug

DNA interactions.

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the lower ability of the cancer cells to repair the damage

introduced by the drugs, which ultimately results in cell

apoptosis. New formulations are being currently used to

enhance the tumor selectivity of anthracyclines using lipo-

some-cotaining complexes.(72,73) The effort currently devoted

to the development of these drugs underscores their en-

ormous potential as anti-tumor agents.

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Review articles

56 BioEssays 27.1