(Received for publication, May 24, 1995; and in revised form, July 26, 1995)
From the
Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage >6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is >2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.
Topoisomerase II is the cellular target for some of the most active agents currently used for the treatment of human cancers(1, 2, 3, 4) . These drugs, which include etoposide, doxorubicin, and mitoxantrone, elicit their antineoplastic effects by a mechanism that is markedly different from that of other enzyme-targeted agents. Rather than acting by inhibiting the catalytic activity of the enzyme, anticancer drugs dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting intermediates in the catalytic cycle of the enzyme(1, 2, 3, 4) . When present at high concentrations, a portion of these transient enzyme-associated DNA breaks are converted to permanent untethered breaks during replication and transcription (5, 6, 7) . Thus, these agents act as poisons and convert topoisomerase II, an essential enzyme, into a physiological toxin that produces protein-linked breaks in the genome of treated cells(1, 2, 3, 4) .
Consistent with the ability to generate DNA breaks, topoisomerase II-targeted drugs produce mutagenic side effects. Insertions, deletions, and chromosomal aberrations have been observed in treated cells and animals(8, 9) . Furthermore, levels of sister chromatid exchange and illegitimate recombination are increased significantly(8, 9) . Finally, 11q23 chromosomal translocations that produce acute myelogenous leukemia have been reported in patients following treatment with etoposide-based regimens(8, 10, 11, 12, 13) .
Approximately 80% of infant leukemias share the same 11q23 chromosomal translocations as are found in the drug-induced leukemias despite the fact that the affected children have never been exposed to anticancer agents(14) . Coupled with the fact that topoisomerase II poisons act by exploiting the natural ability of the enzyme to cleave DNA, this finding raises two possibilities. First, there may be cellular factors that induce topoisomerase II-mediated DNA breakage in vivo and trigger DNA recombination, mutagenesis, or cell death pathways. Second, anticancer agents targeted to this enzyme may actually represent exogenous counterparts of these factors.
Since anticancer agents presumably act at the topoisomerase
II/nucleic acid
interface(1, 2, 3, 4, 15) ,
we questioned whether specific lesions generated by spontaneous DNA
damage function as these endogenous cleavage-enhancing factors. Abasic
(apurinic/apyrimidinic) sites were chosen for this study because they
are the most commonly formed endogenous lesion in DNA
(10,000/mammalian
cell/day)(16, 17, 18, 19) . Results
indicate that abasic sites are potent enhancers of double-stranded DNA
cleavage mediated by Drosophila melanogaster topoisomerase II.
This suggests that abasic sites represent cellular topoisomerase II
poisons and implies that anticancer drugs mimic the actions of these
endogenous DNA lesions.
Topoisomerase II was purified from D. melanogaster embryonic Kc cells as described by Shelton et
al.(20) . Negatively supercoiled pBR322 plasmid DNA was
prepared as described previously(21) . Tris was obtained from
Sigma, proteinase K and SDS were from Merck, topoisomerase I was from
Life Technologies, Inc., and [-
P]ATP
(
3000 Ci/mmol) was from Amersham. Bacteriophage T4 endonuclease V
was prepared by the procedure of Lloyd et al.(22) and
was the generous gift of Dr. R. S. Lloyd (The University of Texas
Medical Branch at Galveston). Etoposide was from Sigma and was prepared
as a 10 mM drug stock solution in 100% dimethyl sulfoxide
prior to use. All other chemicals were analytical reagent grade.
The presence of abasic sites in pBR322 DNA stimulated double-stranded DNA cleavage mediated by D. melanogaster topoisomerase II ( Fig. 1and Fig. 2, inset). Cleavage was increased nearly 4-fold in plasmids that contained a single abasic site (statistical average) and more than 6-fold in plasmids that contained 3 lesions. At 10 nM plasmid/assay, 3 lesions corresponds to an abasic site concentration of 30 nM.
Figure 1: Enhancement of topoisomerase II-mediated double-stranded DNA cleavage by abasic sites. Data represent the averages of two independent experiments. Standard deviations are indicated by error bars. Inset, comparison of double-stranded DNA cleavage levels induced by abasic sites and etoposide. Data represent the averages of three independent experiments. Standard deviations are indicated by error bars. Assays utilized control pBR322 DNA containing 0 abasic sites (None), abasic plasmid containing 3 lesions/molecule (30 nM total abasic sites) (AP), or control DNA plus 100 µM etoposide (Etop).
Figure 2:
Cleavage of DNA containing abasic sites
is mediated by topoisomerase II. Data represent the averages of 3
independent experiments. Standard deviations are indicated by error
bars. DNA cleavage assays utilized control pBR322 DNA containing 0
abasic sites (None), abasic plasmid containing 3
lesions/molecule (AP), or abasic plasmid with the following
modifications: reaction products were not digested with proteinase K (-ProK), samples were treated with 500 mM NaCl (+Salt) or 15 mM EDTA (+EDTA) for
1 min prior to trapping DNA cleavage products with SDS, or reactions
were carried out in the absence of Mg (-Mg
) or topoisomerase II (-Topo II). Inset, an ethidium bromide-stained
agarose gel of DNA cleavage products is shown. Reactions utilized
either control DNA (AP -) or abasic plasmid (AP
+) in the presence or absence of topoisomerase II (TII). The positions of negatively supercoiled (FI),
nicked (FII), and linear (FIII) plasmid are
indicated.
All DNA cleavage assays shown were carried out in the absence of ATP and hence represent cleavage/religation equilibria established prior to strand passage(26) . Abasic sites doubled levels of topoisomerase II-mediated DNA cleavage in the presence of a nonhydrolyzable ATP analog. Therefore, these lesions also enhance DNA cleavage that occurs in the ATP-bound form (i.e. post-strand passage conformation) of the enzyme.
To contrast the potency of abasic sites with that of
a topoisomerase II-targeted anticancer agent, 100 µM etoposide (the most widely prescribed anticancer drug in clinical
use(12, 13) ) was required to produce an equivalent
level of DNA cleavage as seen in the presence of 3 lesions/plasmid (Fig. 1, inset). As extrapolated from the concentration
of abasic sites required to produce one-half maximal cleavage
enhancement (10 nM abasic sites (Fig. 1) versus >25 µM etoposide(27, 28) ), this DNA lesion appears to
be at least 2,000 times more potent than etoposide under the conditions
employed.
A number of control experiments were performed to ensure
that the bulk of cleavage induced by abasic sites was due to the action
of topoisomerase II rather than by chemical degradation or by a
potential contaminating AP endonuclease (Fig. 2). First,
cleavage required the presence of both topoisomerase II and
Mg. Second, the majority of double-stranded breaks
were protein-linked (i.e. linear DNA molecules were not
released unless samples were digested with proteinase K) and were
readily reversed by the addition of salt or by chelation of the
required divalent cation by EDTA. These characteristics are hallmarks
of topoisomerase II-mediated DNA cleavage (29, 30, 31) and are incompatible with the
mechanism of action of AP endonucleases(32, 33) . A
small proportion of double-stranded DNA breaks observed were not
protein-associated. This cleavage may reflect the inherent instability
of abasic sites (16, 17, 18, 19) (as
evidenced by the enzyme-independent increase in nicked (FII) DNA seen
in Fig. 2, inset), which may be exacerbated by exposure
to the large number of nucleophilic aminoacyl residues in topoisomerase
II(34) . Since this later class of breaks may not reflect
events mediated by the active-site tyrosine of topoisomerase II, for
all experiments shown (other than those in Fig. 2),
double-stranded DNA breaks that were not protein-associated (i.e. plasmid molecules that were not digested with proteinase K, but
migrated with the electrophoretic mobility of free linear pBR322 DNA)
were subtracted as background from the total double-stranded cleavage
observed.
As determined by electrophoresis in agarose gels
containing the intercalating agent, chloroquine, the average linking
number of pBR322 was not altered significantly by the presence of 3
abasic sites/plasmid. Therefore, the enhancement of topoisomerase
II-mediated DNA cleavage by abasic sites was not due to a global change
in plasmid topology. Furthermore, abasic sites stimulated cleavage in
linear DNA substrates, ()indicating that superhelicity is
not needed for cleavage enhancement. Finally, the effect of abasic
sites appears to be specific for type II topoisomerases, as no cleavage
stimulation was observed in the presence of topoisomerase I.
Topoisomerase II establishes a DNA cleavage/religation equilibrium within a few seconds when control pBR322 molecules are used as substrates (35) (Fig. 3). This rapid attainment of equilibrium probably reflects the plethora of potential recognition/cleavage sites that are available to the enzyme in the plasmid. In contrast, cleavage of pBR322 that contained only 3 abasic sites was time-dependent and required 6 min to achieve maximal cleavage (Fig. 3). Beyond 6 min, levels of cleavage remained stable. This indicates that topoisomerase II does not initially bind its DNA substrate at abasic sites and suggests that it locates these lesions by a scanning mechanism. If this is the case, the enzyme may be able to locate isolated abasic sites, even if they are separated by long regions of DNA.
Figure 3: Time course of topoisomerase II-mediated DNA cleavage. Data represent the averages of two independent experiments. Standard deviations are indicated by error bars. DNA cleavage reactions were incubated for the indicated times and utilized DNA containing 0 (None) or 3 (Abasic) abasic sites/plasmid.
Topoisomerase II-targeted anticancer agents enhance enzyme-mediated DNA breakage by one of two mechanisms(1) . Drugs such as etoposide (Fig. 4) and amsacrine act primarily by inhibiting the ability of topoisomerase II to religate its cleaved DNA substrate. Alternatively, agents such as quinolones, genistein, nitroimidazoles, pyrimidobenzimidazoles, and ellipticine have little effect on religation and presumably act by enhancing the forward rate of cleavage(1, 36, 37) . Abasic sites did not inhibit the apparent first order rate of DNA religation (Fig. 4). In fact, the religation rate of plasmid molecules containing 3 abasic sites was nearly twice that of control DNA. This finding provides strong evidence that abasic sites enhance nucleic acid breakage primarily by increasing the forward rate of cleavage.
Figure 4: Effect of abasic sites on topoisomerase II-mediated religation of cleaved DNA. Data represent the averages of two independent experiments. DNA religation was initiated by shifting the temperature from 30 °C to 55 °C and was stopped at the indicated times. At time = 0, levels of double-stranded DNA cleavage were set to 100%. Reactions utilized plasmid containing either 0 (None) or 3 abasic sites (Abasic), or 0 abasic sites plus 100 µM etoposide (Etop).
Although the cytotoxic potential of topoisomerase II poisons
reflects their ability to enhance DNA breakage, these agents also
inhibit the catalytic strand passage reaction of the
enzyme(38) . This was not the case for DNA containing abasic
sites (Fig. 5A). At a catalytic enzyme to plasmid ratio (i.e. 1:3), topoisomerase II relaxed negatively
supercoiled abasic DNA at a rate that was
25% faster than that of
control plasmid. It is notable, however, that catalytic DNA relaxation
is limited by the rate at which the enzyme releases its fully relaxed
DNA substrate(39) . This slow dissociation rate could
potentially mask the stimulatory effects of abasic sites on DNA
cleavage or other reaction steps prior to enzyme recycling. Therefore,
the experiment was repeated utilizing a stoichiometric ratio of enzyme
to plasmid (i.e.
1.3:1) (Fig. 5B). Under
these conditions, which do not require enzyme dissociation, negatively
supercoiled substrates containing 3 abasic sites were relaxed with an
initial velocity that was
2.5 times higher than that of control
molecules.
Figure 5:
Stimulation of topoisomerase II-catalyzed
relaxation of negatively supercoiled plasmid by abasic sites. Data
represent the averages of two independent experiments. Standard
deviations are indicated by error bars. In A,
reactions were carried out using catalytic levels of topoisomerase II
(enzyme:plasmid 1:3). In B, reactions were carried out
using stoichiometric levels of topoisomerase II (enzyme:plasmid
1.3:1).
The enhancement of DNA relaxation by abasic sites is in marked contrast to the inhibitory effects of anticancer drugs, even those that stimulate the forward rate of enzyme-mediated DNA cleavage(1) . However, in all cases examined, these drugs impaired interactions between topoisomerase II and its ATP cofactor (24) (which is required for DNA strand passage(29, 30, 31) ). In contrast, abasic sites did not effect the rate of enzyme-catalyzed ATP hydrolysis (Fig. 6). This lack of inhibition may explain why abasic sites, which enhance the forward rate of cleavage, are able to stimulate the catalytic activity of the enzyme.
Figure 6: Effect of abasic sites on topoisomerase II-catalyzed ATP hydrolysis. Data represent the averages of two independent experiments. Reactions were carried out in the presence of DNA containing 0 (open circles, None) or 3 (closed circles, Abasic) abasic sites/plasmid.
Abasic sites are the first endogenous DNA lesion or modification found to increase topoisomerase II-mediated DNA cleavage. Previous studies examined the effects of ultraviolet-induced cyclobutane pyrimidine dimers (21) or DNA methylation (40) on topoisomerase II activity. Pyrimidine dimers had no effect on DNA cleavage, but rather inhibited overall activity by specifically inhibiting the DNA strand passage step of the topoisomerase II catalytic cycle. Furthermore, methylation suppressed DNA cleavage up to 7 bases from the modified cytosine.
Abasic sites are produced in vivo by a myriad of DNA damaging events. Among these are oxidation (including attack by NO produced as part of the macrophage immune response), ionizing radiation, DNA-reactive chemicals (including alkylating agents used for the treatment of human cancers), and spontaneous hydrolysis (17, 19, 33, 41) . Similar to topoisomerase II-targeted agents, these events induce mutagenesis, recombination, and under excessive conditions, trigger pathways that lead to cell death. Due to the rapid removal of abasic sites by the cellular repair machinery(17, 19) , physiological interactions with topoisomerase II under normal conditions probably are rare. However, the results of the present study indicate that when these interactions occur, they may lead to the production of protein-associated double-stranded DNA breaks. Furthermore, the potential for these interactions are increased under conditions that induce DNA damage or compromise repair systems(42) .
On the basis of the studies presented above, it is proposed that abasic sites are a conduit for numerous cellular insults that ultimately enhance topoisomerase II-mediated double-stranded DNA cleavage. Although DNA scissions produced by this process are transient in nature, they are frequently converted to permanent double-stranded breaks during replication and transcription(5, 6, 7) . Unlike the readily repaired single-stranded DNA nicks normally produced at abasic sites by the actions of AP endonucleases, lyases, and spontaneous elimination reactions(17, 19) , double-stranded breaks are potential hot spots for genomic alterations, chromosomal translocations, and the induction of apoptotic events(8, 9) . This suggests that anticancer agents targeted to the enzyme exert their mutagenic and antineoplastic effects by utilizing a mechanism that mimics the cleavage-enhancing actions of endogenous DNA lesions, thus allowing these drugs to exploit existing cellular pathways.