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1
* University of Tennessee and
St. Jude Children’s Research Hospital, Memphis 38105-2794, Tennessee, USA
1Correspondence: Department of Pharmaceutical Science, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794, USA. E-mail: mary.relling{at}stjude.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: thioguanylated oligodeoxyribonucleotides • etoposide • thiopurine • leukemogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. During the breakage event, topoisomerase II forms an
intermediate complex with DNA substrate in which enzyme is covalently
linked to the 5'-end of cleaved DNA (3
, 4)
. Topoisomerase
II is particularly reactive toward purine-pyrimidine repeat sequences
(5)
. Some anticancer agents, e.g., etoposide, inhibit the
rejoining steps during topoisomerase processing, resulting in
accumulation of covalent DNA-protein complexes, which are reversible
after drug removal (6
, 7)
.
Although interference with topoisomerase II is the primary mechanism
underlying the desired cytotoxic effects of etoposide, this mechanism
has also been implicated in the undesired leukemogenesis associated
with etoposide and other agents that interfere with topoisomerase II
(8
9
10
11
12
13)
. Topoisomerase II agent-related acute myeloid
leukemia carries an almost uniformly fatal prognosis and occurs with up
to a 10% cumulative incidence (14)
. Thus, the utility of
topoisomerase II active agents as anticancer drugs has been compromised
by their leukemogenic properties. Identifying therapy-related risk
factors for etoposide-induced acute myeloid leukemia should allow for
the development of less leukemogenic etoposide-containing regimens.
Many clinical studies with high incidences of secondary leukemia have
included thiopurine therapy preceding or concurrent with etoposide
therapy (14
, 15)
, and we and others (16
, 17)
have shown that the risk of therapy-related leukemia is more common
among patients with a genetic defect in thiopurine methyltransferase or
high thioguanine nucleotide active metabolites. However, the mechanism
by which thiopurines might contribute to etoposide-induced
leukemogenesis remains to be elucidated.
Thiopurines (e.g., mercaptopurine, thioguanine, and azathioprine) are
anticancer and immunosuppressive agents that are anabolized to
thioguanine nucleotides and exert cytotoxic effects via incorporation
of deoxythioguanosine into DNA. These deoxythioguanosine nucleotides
are detectable in DNA of leukocytes after low-dose thiopurine therapy
(18
, 19)
. The pharmacological and biochemical consequences
of such incorporation are unknown, however. Because single mutational
events (e.g., generation of apurinic or apyrimidinic sites or the
deamination of cytosine residues) can act as poisons of topoisomerase
II (20
, 21)
, we hypothesized that deoxythioguanosine
substitution could likewise affect topoisomerase II cleavage activity,
thereby providing a possible mechanistic link between thiopurine
therapy and acute myeloid leukemia following topoisomerase II active
agents.
To gain further insight into whether thiopurines could alter the interaction of topoisomerase II-directed agents with topoisomerase II and DNA, we determined whether deoxythioguanosine substitutions qualitatively or quantitatively altered DNA cleavage produced by human DNA topoisomerase II in the presence and absence of etoposide.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[
-32P]ATP (
6000 Ci/mmol) was from
Amersham Pharmacia Biotech (Piscataway, N.J.); RTS T4 Kinase Labeling
System was obtained from Gibco BRL (Rockville, Md.). Oligonucleotide
sizing markers were from Amersham Pharmacia Biotech (Piscataway). All
other chemicals were of analytical reagent grade.
Wild-type human topoisomerase II was overexpressed using pXY113GalhTOP2
and purified to homogeneity by a modification of the procedure of
Worland and Wang (22)
as described (see ref
23
).
Preparation of modified and nonmodified oligodeoxyribonucleotides
Four 40-base single-stranded oligodeoxyribonucleotides
were synthesized using standard protocols with an automated synthesizer
(Applied Biosystem 380B, Foster City, Calif.), with the wild-type
sequence identical to one previously demonstrated to possess a strong
human topoisomerase II cleavage site (21
, 24)
. We
substituted all three guanosines in the four base pair overhang region
and one immediate 5' to the overhang (one at a time) with thioguanosine
as indicated below:
5'-d(TGAAATCTAACAATGSCGCTCATCGTCATCCTCGGCACCCGT)-3' (-1) top;
5'-d(TGAAATCTAACAATGCGSCTCATCGTCATCCTCGGCACCCGT)-3' (+2) top;
5'-d(ACGGGTGCCGAGGATGACGATGAGSCGCATTGTTAGATTTCA)-3' (+4) bottom;
5'-d(ACGGGTGCCGAGGATGACGATGAGCGSCATTGTTAGATTTCA)-3' (+2) bottom.
A single 6-deoxythioguanosine (GS) was inserted at the indicated positions bracketing and within the cleavage and topoisomerase II site, using standard phosphoramidite chemistry with S6-DNP-dG-CE phosphoramidite, and isolated after a deblocking step by polyacrylamide gel-electrophoresis. The purity of the oligonucleotides by electrophoresis was estimated to be 95%. The presence of 6-thiodeoxyguanosine residue was confirmed by UV spectroscopy (maximum absorption at 340 nm) of the purified oligonucleotide.
Oligonucleotide concentrations were determined spectrophotometrically.
The following molar extinction coefficients
(
260) were used for nucleotides: pA, 15400;
pT, 9300; pC, 7300; and pG, 11700. The molar extinction coefficient for
pGS(340) was 24,800
(25)
.
Prior to labeling, the single-stranded oligonucleotides were purified
by electrophoresis in 7.5 M urea, 12% polyacrylamide. DNA bands were
excised from gels, extracted overnight (21°C) with buffer containing
250 mM sodium acetate (pH 5.2), 50 mM sodium chloride, 1 mM EDTA, and
1% 2 mercaptoethanol, and precipitated by ethanol at -20°C
overnight. The purified oligonucleotides were radioactively labeled at
their 5' termini using the RTS T4 kinase labeling system. Each reaction
mixture contained 15 pmol of oligonucleotide, 10 units of
polynucleotide kinase, and 8 pmol of [-32P]
ATP in a total of 20 µl kinase forward reaction buffer. The labeled
oligonucleotides were further purified prior to the topoisomerase
II-mediated DNA cleavage reaction by electrophoresis as described
below.
Duplex DNA substrates were prepared by mixing equimolar amounts
of radiolabeled and unlabeled complementary strands to a final
concentration of 0.5 pmol/µl and a final specific activity of
2 x 104 cpm/pmol in 10 mM Tris-HCl, pH
8.0. Oligonucleotide concentrations were measured while single
stranded; the stability of double-stranded deoxythioguanosine
containing oligomers at these temperatures has been demonstrated
previously (26)
. Oligonucleotides were heated to 30°C
for 30 min and allowed to anneal overnight at 4°C. A total of 10
[32P-5']-labeled duplex substrates were
prepared (Table 1
): the wild-type (wt) and four modified oligonucleotides (-1 top; +2
top; +2 bottom; +4 bottom) were each prepared with the 5'-end of either
the top or bottom strand labeled.
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Topoisomerase II-mediated DNA cleavage
Cleavage reaction mixtures consisted of 10 mM HEPES-HCl pH 7.9,
0.1 mM EDTA, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1
mM ATP, 2 pmol oligonucleotide, and 2 µl of 1 mM etoposide (or the
solvent 10% DMSO in water). Reactions were initiated by adding 2 µl
of water (negative controls) or 2 µl of 0.255 µg/µl topoisomerase
II, which was diluted with H2O immediately before
use from a concentrated enzyme stock (0.9 to 11.8 µg/µl in a
storage buffer of 50 mM Tris-HCl pH 7.7, 200 mM KCl, 1 mM EDTA, 30%
glycerol, and 0.5 mM dithiothreitol). The total reaction volume was 20
µl. The final ratio of topoisomerase II to DNA was 1.5 pmol per 2
pmol of DNA substrate, similar to that previously described
(21)
. The incubation time for the cleavage reaction and
concentration of etoposide were optimized using the wild-type DNA
substrate in preliminary experiments assessing cleavage at 0 to 45 min
and at zero, 5, 10, 50, and 100 µM etoposide.
Reactions were incubated at 30°C for 3–4 min, then stopped by transferring to ice and addition of 2 µl 10% sodium dodecyl sulfate, followed by 1.5 µl of 250 mM EDTA. Cleavage products were digested with proteinase K (2 µl of a 0.8 mg/ml solution) for 20 min at 37°C, coprecipitated with 1 µl of 5 µg/µl tRNA by ethanol, and resuspended in 3 µl of loading dye (10 ml of formamide, 10 mg of bromphenol blue, and 200 µl of 0.5 M EDTA, pH 8.0). Products were resolved by electrophoresis in denaturing 7.5 M urea, 12% polyacrylamide sequencing gel in 100 mM Tris-borate-2 mM EDTA buffer, pH 8.3. Gels were dried without fixing and the cleavage products were visualized using a Molecular Dynamics PhosphorImager system. The amounts of cleavage products (the 15 or 21 mers observed with top vs. bottom 5' labeling, respectively) and the parent duplex (40 mer) were calculated using the volume quantitation subroutine of ImageQuaNT Software (Molecular Dynamics Inc., Sunnyvale, Calif.) which estimates the integrated intensity of all the pixels in an equal size rectangle drawn around the band. Cleavage (defined as the ratio of the intensity of the cleavage product to the starting oligonucleotide) in each experiment was expressed relative to the wild-type substrate in the presence of topoisomerase II (which was assigned a value of 1). The average ratio of product to starting oligonucleotide density for the wild-type substrate without etoposide was 0.088% (standard error=0.013%). Topoisomerase II-induced cleavage was assessed in 2–3 independent experiments for each modified oligonucleotide, each of which included the oligonucleotide labeled (separately) on the top and bottom strands such that all assessments of cleavage represent the mean of at least four values. For statistical analyses only, the variability in cleavage across experiments for the wild-type substrate in the absence of etoposide was assumed equivalent to the variability in cleavage in the presence of etoposide. Unpaired t tests were used to assess statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As shown
in Fig. 1
and Fig. 2
, deoxythioguanosine substitutions at positions +4 bottom and -1 top
did not qualitatively change the position of the topoisomerase
II-mediated cleavage site relative to the wild-type substrate, with
labeling of the top vs. bottom strands always producing 15 mer or 21
mer cleavage products, respectively. Qualitative changes in the
position of cleavage were not noted for deoxythioguanosine
substitutions at the +2 top or +2 bottom positions either (data not
shown). However, there were quantitative effects of deoxythioguanosine
substitution on the topoisomerase II scission products (Fig. 3
). The internal sites at +4 bottom and +2 top stimulated DNA cleavage
two- to threefold relative to wild-type substrate (P<0.001
and P=0.008, respectively), whereas the -1 upper
substitution inhibited DNA cleavage almost twofold
(P=0.011), and the +2 bottom substitution had no effect on
cleavage (P=0.51) (Fig. 3)
.
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Etoposide enhanced the topoisomerase-II mediated DNA cleavage of the
wild-type substrate by 6-fold and enhanced cleavage of each of the
four thioguanosine-substituted oligonucleotides by 1.8- to 9.7-fold
(Fig. 3)
. The +4 bottom substitution resulted in accumulation of higher
levels of cleavage products than were observed with the wild-type
substrate in the presence of etoposide (P=0.015). Thus, the
position of deoxythioguanosine-substitution influenced the potency of
topoisomerase II-induced DNA cleavage: the duplex containing
modification at +4 position on the bottom strand demonstrated the
greatest amount of cleavage products, with or without etoposide, and
-1 position on the top strand was associated with lower levels of
topoisomerase II-mediated DNA cleavage products.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 24
, 27)
affect
topoisomerase II-mediated DNA cleavage has been sparked by the
possibility that such DNA alterations contribute to the leukemogenic
properties of topoisomerase II active agents. Here we have demonstrated
that incorporation of deoxythioguanosine, a DNA alteration known to
occur in normal human hematopoietic cells after thiopurine therapy
(18
, 19)
affects the potency of topoisomerase II-mediated
DNA cleavage in the absence and presence of etoposide. These findings
may have particular significance in that increased exposure to
thioguanine nucleotide metabolites has been linked to the risk of
topoisomerase II agent associated (16)
and other
therapy-related myeloid leukemias (17)
and secondary brain
tumors (28)
.
Depending on the position of deoxythioguanosine substitution,
either enhanced or diminished topoisomerase II-mediated cleavage was
observed. It is possible that either effect could contribute to
leukemogenesis. The relationship between dosage of topoisomerase II
active agents and the risk of topoisomerase II agent-associated acute
myeloid leukemia is not clear (10
, 29
)
. In fact, some
very dose-intensive etoposide-containing regimens are only weakly
leukemogenic (14
, 29
, 30)
, whereas some relatively
low-dose regimens are leukemogenic (31
32
33)
. We
hypothesize that a very potent interference with topoisomerase II will
result in cell death (and thus less leukemogenesis), whereas somewhat
less potent interference with topoisomerase II might result in more
surviving cells with sublethal nonhomologous recombination, thereby
creating the opportunity for a leukemogenic rearrangement.
Whereas thioguanine incorporation into DNA and alteration of the
potency of topoisomerase II-mediated DNA cleavage is a reasonable
mechanism by which thiopurines could enhance the effects of
topoisomerase II targeting agents, thiopurines also produce other
effects that could be leukemogenic. Thiopurines can alter endogenous
nucleotide pools (and thereby further affect topoisomerase II
complexes) (34)
and deplete S-adenosylmethionine (and
thereby alter DNA methylation), and thioguanine DNA incorporation can
affect other DNA-targeted enzymes (e.g., mismatch repair enzymes;
35
, 36
). Of course, all of these mechanisms have the
potential to contribute to the desired cytotoxic effects of
thiopurines, as well as to their leukemogenic or carcinogenic effects.
The position-dependent effects of deoxythioguanosine
substitution are similar to those observed with abasic sites and
mismatches (24
, 27)
. In the absence of etoposide,
deoxythioguanosine enhanced topoisomerase II cleavage at positions +2
top and +4 bottom relative to the wild-type substrate. The
topoisomerase II-catalyzed reaction can be considered as two
consecutive steps: 1) transesterification, leading to
cleavage of the phosphodiester bond and formation of a new
phosphodiester bond with the hydroxyl group of a tyrosine residue in
topoisomerase II; and 2) religation of the original
phosphodiester bond. Modification of DNA by deoxythioguanosine inserts
resulting in structural distortions of B-form DNA could change the
interaction between DNA and topoisomerase II by altering the formation
of the phosphotyrosine linkage between enzyme and DNA or by affecting
the religation reaction (Fig. 4
).
|
In the absence of etoposide, the greatest inhibition of cleavage was
observed with deoxythioguanosine at the -1 top position, which is
closest to the site of cleavage (Fig. 4A
). Similar
inhibitory effects were observed when apurinic sites were introduced at
the -1 position (24)
, suggesting that changes at -1 may
alter the geometry of the phosphodiester bond/tyrosine interaction.
Although addition of etoposide results in greater cleavage with the -1
top oligomer (as it does for all of the substrates), the level of
cleavage remains significantly lower than that observed with wild-type
substrate plus etoposide, indicating the overriding importance of the
deoxythioguanosine substitution at -1.
In contrast, greater cleavage was observed when deoxythioguanosine was
inserted opposite the cleaved phosphodiester bond at the +4 bottom
position both in the absence and presence of etoposide (Table 1
, Fig. 3
). Again, these results are comparable to those observed with an
apurinic site at the identical position (21)
. This result
is consistent with a mechanism whereby religation of the broken DNA
strands is hindered by structural alterations of Watson-Crick
complementarity at the 5' terminus due to deoxythioguanosine
modification at +4 in the bottom strand (Fig. 4B
) and might
mimic the action of topoisomerase II inhibitors. Similar interference
with the ligation of thioguanylated DNA by T4 DNA ligase was observed
(37)
.
Our results indicate that thiopurine treatment is likely to change the efficacy of topoisomerase II-mediated DNA cleavage, both in the absence and presence of topoisomerase II active agents. Deoxythioguanosine insertions might create hot spots of recombination due to alteration of the critical religation step catalyzed by topoisomerase II. Such recombinogenic events, while potentially contributing to the desired antitumor effects of thiopurines (with or without topoisomerase II inhibitors), could also contribute to undesired leukemogenic effects of thiopurine therapy and would be consistent with recent clinical observations.
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ACKNOWLEDGMENTS |
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Received for publication February 21, 2000.
Revision received May 5, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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