From the Department of Biology, Indiana University, Bloomington, Indiana 47405
Received for publication, July 18, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Loss of the human DNA mismatch repair pathway
confers cross-resistance to structurally unrelated anticancer drugs.
Examples include cisplatin, doxorubicin (adriamycin), and specific
alkylating agents. We focused on defining the molecular events that
link adriamycin to mismatch repair-dependent drug
resistance because adriamycin, unlike drugs that covalently modify DNA,
can interact reversibly with DNA. We found that adriamycin,
nogalamycin, and actinomycin D comprise a class of drugs that
reversibly inhibits human mismatch repair in vitro at low
micromolar concentrations. The substrate DNA was not covalently
modified by adriamycin treatment in a way that prevents repair, and the
inhibition was independent of the number of intercalation sites
separating the mismatch and the DNA nick used to direct repair, from 10 to 808 base pairs. Over the broad concentration range tested,
there was no evidence for recognition of intercalated adriamycin by
MutS Recent studies suggest that the human mismatch repair
(MMR)1 pathway contributes to
the cytotoxic effect elicited by several different anti-neoplastic
agents. MMR normally functions to correct base misincorporations that
arise during replication, but it may also be involved in pathways that
react to specific types of DNA injury (reviewed in Refs. 1-6).
Cultured cells with low level resistance to cisplatin, DNA alkylating
drugs, 6-thioguanine, and doxorubicin (adriamycin (AD)) have been
identified with deficiencies in human MMR proteins (reviewed in Refs. 3
and 7-9). In response to cisplatin
(cis-diamminedichloroplatinum(II)) or alkylating agents such
as
N-methyl-N'-nitro-N-nitrosoguanidine,
N-methyl-N-nitrosourea, and temezolomide, the
tumor suppressor p53 becomes stabilized in MMR proficient but not in
MMR-deficient cell lines, implying that there is
lesion-dependent communication between MMR and a p53-dependent apoptotic pathway (10-13). It has also been
shown that sensitivity to the alkylating agent
N-methyl-N'-nitro-N-nitrosoguanidine can exist in the absence of p53 in some cell lines yet still depend on
a functional MMR pathway (13). In addition, mediation of cell cycle
arrest or cell death subsequent to cisplatin exposure may involve MMR
and the tumor suppressor p73, a p53-independent pathway (14). It may be
that MMR functions as a "damage sensor" in this respect; it can
recognize DNA lesions and initiate a series of events that result in
cell cycle arrest and death (15-18). O'Driscoll et al.
(19) have argued that such damage sensing may be confined to specific
types of lesions rather than operating as a general sensor of damage.
Two prominent models have been proposed to explain the connection
between the MMR pathway and the cytotoxic effects of specific anticancer drugs. The futile repair model (20-22) posits that
repeated, failed attempts at removal of DNA lesions can lead to
apoptosis. In this model, DNA lesions are recognized and processed by
the MMR pathway, but because MMR excises a tract from the newly
incorporated strand, damage in the parental strand is not removed.
Repeated processing attempts create persistent gaps that may trigger
cell cycle arrest or cell death. Alternatively, a more direct signaling pathway could be responsible. Assembly of repair factors at the site of
a lesion may trigger events that lead to apoptosis (16-18). This could
include binding of MutS The primary role of hMutS The futile repair and direct signaling models both depend on
recognition of covalent DNA modifications. However, AD differs from
drugs such as cisplatin and DNA alkylators because AD can reversibly
bind DNA. Because this is a distinguishing feature among these
anticancer drugs, we examined the effect of AD on the MMR pathway with
the goal of defining the early steps in the MMR-mediated response to
AD. By using an in vitro repair assay for human MMR activity
(29), we have found that AD, nogalamycin, and actinomycin D inhibit the
correction of DNA mismatches in vitro. We have exploited
this assay to distinguish among several possible models that seek to
explain how these drugs might interact with MMR to produce a cytotoxic response.
Chemical Reagents--
All reagents, including those required
for the human MMR assay and band shift experiments, were purchased from
the Sigma. Stock solutions of daunomycin were prepared in water,
whereas AD and actinomycin D were prepared in a buffer containing 25 mM Hepes-KOH (pH 7.5) and 110 mM KCl (buffer
A). Etoposide (100 mM) and m-amsacrine (5 mM) were prepared in 100% Me2SO and
working concentrations were made by diluting the Me2SO
stock into buffer A. Nogalamycin was dissolved in 75%
Me2SO and diluted into buffer A. Whenever Me2SO
was used as a solvent, the final concentration did not exceed 2% in
the assay, a level that does affect MMR in vitro (data not
presented). Concentrations of AD, daunomycin, nogalamycin, actinomycin
D, and ethidium bromide were determined spectroscopically using the
following extinction coefficients: AD, Mismatch Repair Substrates--
G·T mispaired substrates with
a single-strand break placed so that 128 bp intervene reading 5' Preparation of Nuclear Extracts and MutS Mismatch Repair Assay--
The MMR assay was performed as
described (29) with the following minor modifications. Reactions
(15 µl) contained 21 fmol of G·T nicked heteroduplex substrate, 45 µg of HeLa extract, 5 mM MgCl2, 110 mM KCl, 1 mM glutathione, 1 mM each
of dNTP, 50 µg/ml bovine serum albumin, and 1.5 mM ATP.
When possible, master mixes were made of all components except the drug
under consideration and HeLa extract. To score for repair, the
recovered substrates were digested with Bsp106 (1 unit/100
ng of DNA) to linearize the substrate and with HindIII (1 unit/100 ng of DNA) to score for G·T mispairs corrected to AT
or XhoI to score A·C corrected to GC, respectively.
Repaired molecules are present as bands of 3.1 and 3.3 kb, the products
of Bsp106 and HindIII or XhoI
digestion. The restricted DNA fragments were separated on a 1% agarose
gel in 1× (Tris-acetate ethylenediamine tetraacetic acid, pH
8.8) for 300 V·h, stained with ethidium bromide and the image
captured using a ChemiImager 4000 cooled CCD camera (Alpha Innotech).
When agarose gels are presented in figures, the information is given as
an inverted image, where the flourescence associated with stained DNA
appears as a dark band on a light background. The NIH image software
(version 1.62) was used to calculate the relative percentage of bands
in a given lane.
Mismatch Repair Assays in the Presence of DNA
Intercalators--
The MMR assays were carried out exactly as
described above, except that the drug of interest and the HeLa extract
were added to the side of a 1.5-ml Eppendorf tube containing all the
assay reagents and placed on ice. Reactions were initiated by pulse centrifugation to combine the components, briefly mixed by manual agitation, and then placed at 37 °C. Similar results were obtained when the drugs were premixed either with the extract or the assay mixture containing the heteroduplex substrate.
Testing for AD-induced Covalent Damage--
Incubations were
carried out in the MMR assay buffer described above using 21 fmol of
G·T heteroduplex with a nick 128 bp 5' of the mispair and performed
in the presence or absence of 10 µM AD. In the pair of
experiments, the DNA substrate and AD were incubated for 20 min at
37 °C in the presence of HeLa nuclear extract as described for the
MMR assay (above). In a parallel mock experiment, the HeLa proteins
were omitted. After incubating the heteroduplex substrate for 20 min at
37 °C in the presence or absence of 10 µM AD, the mock
treated substrates (plus or minus AD) were precipitated from the assay
by adding sodium acetate to 0.3 M and 2.5 volumes of
absolute ethanol. The samples were centrifuged for 30 min at
14,000 × g, and the bulk of the AD remained in the
supernatant, allowing recovery of the DNA. The precipitated substrates
were then washed with absolute ethanol, air dried, and assayed under
standard conditions with HeLa nuclear extract to test for the presence
of covalent modifications that might inhibit mismatch correction.
Mobility Shift Assays using MutS
Band shift experiments were performed with 4 fmol of labeled G·T
heteroduplex or AT homoduplex oligonucleotide and 200 fmol of unlabeled
AT homoduplex oligonucleotide competitor. Reactions were carried out on
ice in a 15-µl volume containing 110 mM KCl, 25 mM Hepes·KOH (pH 7.5), 50 µg/ml bovine serum albumin,
100 ng of (400 fmol) purified MutS Our goal in this work was to establish how the anticancer agent AD
interacts with the human MMR pathway at the molecular level. The
biological basis for this putative interaction derives from the
observation that human cells resistant to cisplatin by virtue of the
loss of mismatch repair are also cross-resistant to other DNA-interacting drugs, a structurally diverse group that includes AD
(10, 34). A direct connection has been established for agents such as
cisplatin or specific DNA alkylating agents, because they generate DNA
lesions that are recognized by MutS AD Blocks an Early Step in MMR--
As a first step, we asked
whether AD is capable of interfering with the repair of a 6.4-kb
circular G·T heteroduplex by HeLa nuclear extract (Fig.
1). The MMR assay is based on the
nick-directed correction of the G·T heteroduplex, where the substrate
DNA is recovered from extracts by SDS treatment, followed by phenol
extraction and precipitation (29). Mismatch correction is scored using two restriction enzymes; Bsp106 linearizes all the molecules
at a site remote from the mismatch, whereas HindIII scores
for nick-directed G·T correction to A·T. DNA bands at 3.1 and 3.3 kb are diagnostic for site-specific mismatch correction. In the absence
of digestion with restriction enzymes, the circular DNA was recovered
as a high molecular weight species that barely enters the 1% agarose gel (see Fig. 1 at 1 µM AD). This species appears to
represent catenated DNA circles, because HeLa nuclear extracts have
been shown to catenate relaxed circular DNA into large networks that can be released upon linearization (35).
Experimentally, two results strongly support the identification of the
high molecular weight species as catenated DNA. First, the putative
catenate yields a single, linear product upon digestion with the unique
cutter Bsp106 (Fig. 1). Second, treatment of the large
molecular weight species with purified type II topoisomerase from
Drosophila (33) yields a relaxed circular species that comigrates with the original
substrate.2 Third, as
described below, the presence of the type II topoisomerase inhibitor
adriamycin prevented formation of the high molecular weight species.
Three related phenomena were apparent when the MMR assay was performed
in the presence of low micromolar concentrations of AD. First, we
observed a sharp decline in repair efficiency when AD was present at
low micromolar concentrations (Fig. 1, panel I). Second, in
the absence of digestion by Bsp106 and HindIII, a
majority of the substrate was recovered from the assay as supercoiled molecules at an AD concentration of 8 µM (Fig. 1,
panel II). The assignment is based on the facts that the DNA
molecule comigrates with supercoiled DNA and that digestion with a
restriction enzyme having a unique site yields a linear product.
Formation of a supercoiled species requires ligation of the nicked
circle into a covalently closed form, and the product is likely to be
positively supercoiled because of ligation in the presence of the
intercalating drug (36). Third, using AD concentrations that inhibit
MMR (e.g. 8 or 30 µM), catenated substrate was
not observed (Fig. 1, panel II). This indicates that
concomitant with MMR inhibition, AD blocked the catenation activity in
the nuclear extract. Furthermore, it shows that inhibition occurred
prior to the long patch strand excision. If AD had inhibited the strand
excision or resynthesis step, a relaxed, gap-containing product would
result instead of supercoiled substrate.
Correcting mispairs requires the coordinated effort of several
independent protein activities prior to strand excision (5). Multiple
steps may therefore be envisioned as sites where AD might interfere
with mismatch recognition or processing (Fig.
2). We have tested five models that
address potential molecular mechanisms for this in vitro
inhibition: 1) MutS AD Does Not Cause DNA Damage That Blocks MMR--
In addition to
reversibly intercalating into DNA, the anthracycline AD can be reduced
in vivo to generate reactive semiquinone species or reduced
oxygen radicals capable of damaging or cross-linking DNA strands
(reviewed in Ref. 38). The cytotoxic action of AD has also been
correlated with its ability to form DNA cross-links in vivo
(39), although similar reactions were not observed in cell free
extracts (39, 40). We therefore asked whether incubation of the
substrate DNA with AD produced DNA damage capable of preventing correction of the G·T mispair.
Heteroduplex substrate was incubated with AD under conditions identical
to the MMR assay, except that HeLa nuclear proteins were absent (see
"Materials and Methods"). The DNA substrate was recovered by
ethanol precipitation, which lowers the AD concentration to levels
undetectable by visible absorbance, well below those having a
measurable effect on MMR. Precipitation is not expected to affect
covalent DNA modifications or cross-links. We found that the G·T
heteroduplex, whether treated with 0 or 10 µM AD and
reisolated by precipitation, was repaired with equal efficiency in each
case (Fig. 1, panel III). Covalent damage that can block repair in vitro, therefore, is not inflicted on the DNA by
AD under the assay conditions in the absence of HeLa proteins.
In parallel experiments, concentrated HeLa nuclear nuclear extract was
incubated with 5 µM AD in the absence of G·T substrate DNA. This experiment was intended to test the possibility that the
extract can reductively activate AD. The mixture was then diluted into
a MMR assay containing substrate DNA to achieve an AD concentration
below that found to substantially inhibit repair (1.3 µM). Under these conditions, the mismatch was corrected
with efficiency similar to untreated HeLa nuclear extract (data not shown). Overall, we found no evidence for covalent modification of MMR
protein components or DNA substrate that contributes to loss of repair
competence. However, we could not exclude the possibility that covalent
DNA modification occurs when all of the assay components are present.
This is because the repair reaction absolutely depends upon the
presence of a single-strand nick to direct repair, and this nick
normally becomes ligated under the assay conditions. Heteroduplex DNA
recovered from an assay is therefore not competent for use in
subsequent assays.
Actinomycin D, Nogalamycin, and Daunomycin Inhibit MMR at
Concentrations Similar to AD--
To explore the importance of DNA
intercalation by AD in the mechanism of inhibition of MMR, other
intercalators were tested for their ability to inhibit MMR in
vitro. The compounds tested can be sorted into three general
classes, the first of which includes AD, nogalamycin, daunomycin, and
actinomycin D. Nogalamycin and daunomycin are anthracyclines that
possess anti-tumor activity and are structurally similar to AD,
containing an intercalating chromophore substituted by a bulky
carbohydrate. Nogalamycin contains an additional carbohydrate
substitution, and intercalation depends on the passage of one bulky
group through the DNA helix (41). Actinomycin D is an inhibitor of
DNA-dependent RNA polymerase (42) and is composed of an
intercalating chromophore with two bulky circular peptides.
Fig. 3 shows that this first class of
drugs inhibits MMR at concentrations from 0 to 5 µM, with
AD nogalamycin and actinomycin D showing close similarity in their
ability to inhibit MMR. The relative ranking of these drugs as MMR
inhibitors is also qualitatively consistent with the DNA binding
affinities of these drugs. For example, daunomycin is the closest
structural analog of AD, and it displays a 2-fold higher
IC50 and a shallower concentration dependence of
inhibition. This observation is consistent with the fact that
daunomycin, when tested for DNA binding affinity under the same
experimental conditions as AD, binds less tightly by a factor that
ranged from 1.5 (43) to 4 (44, 45). This is the only case where
pairwise comparisons of binding affinities can be made reliably between
the drugs tested and AD. Nonetheless, the dissociation constants
determined for AD (38) and actinomycin D (46) are essentially
identical, as are their ability to inhibit MMR. No comparable value for
nogalamycin has been reported, although its intercalation gives
sequence-dependent differences in DNase I footprints when
present at low micromolar concentrations (47).
Ethidium bromide is a prototype intercalator that lacks bulky
substitution, and it represents the second class of inhibitors. When
tested in a MMR assay (Fig. 3), two distinguishing features were
observed. First, the IC50 for ethidium bromide was roughly 10-fold higher than the first class of drugs, and second, the slope of
the inhibition through the central data points was roughly 6-fold
reduced. The reduced ability of ethidium bromide to inhibit MMR, in
comparison with AD, also parallels its reduced binding affinity for
DNA. When compared directly, ethidium bromide bound 7-fold less well to
pBR322 DNA than AD (48). Although association constants for ethidium
bromide binding to DNA are variable, the measured binding affinities
are consistent with a roughly 10-fold reduced affinity for DNA when
compared with AD (49, 50). However, depending on the substrate DNA,
conditions, and experimental approach, the measured affinity of
ethidium bromide for DNA was variable (for example, see Refs. 51 and
52).
Structurally Distinct Topoisomerase Inhibitors Do Not Inhibit Human
MMR--
Because AD, actinomycin D, and nogalamycin are all known to
inhibit topoisomerase activity, we asked whether other inhibitors of
either type I or type II topoisomerases could inhibit DNA MMR. This is
critical in light of the observation that MMR inhibition occurs
concurrently with a block to substrate catenation, implicating inhibition of a type II topoisomerase activity. We tested the type II
topoisomerase inhibitors etoposide and m-amsacrine for inhibition of MMR, as well as the type I toposimerase inhibitor camptothecin, and the prokaryotic gyrase inhibitor novobiocin. None of
the topoisomerase-blocking drugs were found to inhibit MMR over the
concentration range tested, up to at least 300 µM in each
case (not shown). In some cases, drug solubility limited testing at
higher concentrations.
Of special note in this group of drugs are etoposide and
m-amsacrine. Etoposide is a nonintercalating inhibitor of
type II topoisomerase (53), but we found no evidence for an effect on mismatch correction in vitro (data not shown).
m-Amsacrine also represents the third class of DNA
intercalating drugs, and it had no effect on MMR (data not shown). The
observation that a DNA intercalating drug is without effect on MMR is
less surprising in light of the fact that m-amsacrine has a
relatively weak association constant for DNA (KA = 4 × 104 M MMR Inhibition Is Independent of the Distance between the Nick and
Mispair--
To test whether AD inhibits repair by physically
restricting the movement of proteins, such as MutS
In the absence of AD, the absolute level of repair was remarkably
similar for the three substrates tested. Approximately 50% of the
A·C heteroduplex molecules were repaired in each of the three
substrates as the distance separating the nick and mismatch varied from
10 to 808 bp. The relative extent of repair was then plotted as a
function of AD concentration (Fig. 4).
The drug concentration needed to block repair was indistinguishable
among the three substrates. Although this experiment does not address
the possibility that MMR inhibition results from intercalation at a
specific site, such as directly at the nick or mismatch, it does not
support the possibility that intercalated chromophores with bulky side groups contribute a physical block to communication between these two
sites. Furthermore, intercalation directly at the nick site is an
unlikely mechanism to explain the observed inhibition, because it does
not prevent nick ligation by the HeLa extract at concentrations that
completely block MMR (Fig. 1).
Traditionally, in vitro assays have been performed on
substrates where the mispair to nick separation ranges roughly from 125 to 1000 bp (29, 54, 55). The A·C substrate, where the nick and
mismatch are separated by only 10 bp, represents a setting where little
information is available about the MMR pathway. Although we have not
yet shown rigorously that repair of the shorter tract substrate is
dependent upon the identical set of proteins required for long patch
MMR excision and resynthesis, mismatch correction is minimally
dependent upon the MSH2 protein. Heteroduplexes with either a 10- or
128-bp separation between nick and mismatch were incubated with LoVo
nuclear extract, which functionally lacks MSH2 protein. When the nick
is 128 bp removed from the mismatch, mismatch correction is
nick-dependent, strand-specific, and dependent upon the
addition of purified MutS
In addition to the complementation studies, other hallmarks of MMR
in vitro are present (29, 55, 57). For example, maximal repair efficiency of roughly 50% was reached within 15 min, and prolonged incubation had little further effect. Although MMR is classically considered a "long patch" pathway, these data are consistent with a mismatch correction activity that can access a strand
signal at substantially shorter distances. We are currently exploring
the physical limits of MMR where the strand discrimination signal is
positioned close to the mismatch. It is also interesting to note that
Gradia et al. (58), using DNase I footprinting, showed that
the MSH2/MSH6 heterodimer protects a 25-bp asymmetric footprint
surrounding the mismatch. Such binding would place the nick 10 bp
removed from the mismatch either within or at the edge of the initial
MutS Intercalated AD Is Not Recognized as a Mismatch by MutS
Because AD intercalation is a stochastic process, it is not possible to
introduce an intercalated base at the same site in each member of the
population of oligonucleotides as it is for a G·T mismatch. The fact
that the 30-mer oligonucleotide can accommodate multiple AD
intercalations, up to an average of one every five base pairs near
saturation (44, 56) must also be taken into account. Based on published
dissociation constants determined for AD intercalation that range from
240 to 435 nM (reviewed in Ref. 38), homoduplex
oligonucleotides were incubated with AD over a 10,000-fold
concentration range, from 1 nM to 10 µM.
These were chosen to ensure that MutS
As expected, MutS
A similar situation was observed for other intercalating MMR inhibitors
tested, which include actinomycin D, nogalamycin, and ethidium bromide.
In each case, binding of MutS
It is essential to note minor differences between MMR inhibition in the
crude nuclear extract and the defined loss of mismatch recognition in
the bandshift assay. First, despite the fact that the bandshift
experiment is performed with a subset of the DNA sequence found in the
MMR assay substrate flanking the G·T mismatch, the DNA content is not
identical when compared with the assay substrate. The drugs tested
display very different binding affinities for DNA and patterns of
cooperativity, depending upon the sequence content and length (43, 44,
56). Second, in the case of the MMR assay, each drug is present
throughout the assay, whereas in the bandshift, the negatively charged
complex and positively charged drug must be diluted with load dye,
introduced into an acrylamide gel, and placed under a constant voltage.
Finally, the crude extract contains an estimated 4 µg of RNA in each
assay (not shown), in comparison with the 80 ng of double-stranded
G·T substrate. The RNA pool provides an undefined repository for
intercalating drug, obscuring our ability to make rigorous comparisons
between the two types of experiments. Although purified tRNA added to the bandshift experiments or the in vitro MMR assay had
minimal effects in either assay, uncharacterized nucleic acids remain a
source of minor differences between the two types of experiments.
Because the inhibition of MMR in vitro occurred at
concentrations similar to those that prevented mispair recognition, we also sought to establish that the binding affinity of AD to the oligonucleotide substrate was comparable with its affinity for bulk
DNA. The fluorescence of AD is quenched upon intercalation into DNA,
and this property has been used to determine DNA binding affinity (59).
The homoduplex oligonucleotides used in the mobility shift assay
yielded a macromolecular dissociation constant of roughly 200 nM (data not shown). This is in good agreement with published values using calf thymus and other oligomeric DNA (38). Although this strengthens the conclusion that AD intercalation disrupts
mismatch recognition, our data do not allow us define the mechanism by
which AD blocks the interaction between MutS In this work, we focused on clarifying the mechanism by which AD
interacts with the human MMR pathway. AD, along with covalent DNA-modifying drugs such as cisplatin or alkylating agents, have the
potential to trigger a cytotoxic response mediated by the MMR pathway.
However, unlike drugs that cause genome-wide covalent DNA damage, AD
has the potential to engage a MMR-dependent apoptotic response without directly modifying DNA. Although AD can be reduced in vivo to generate reactive oxygen species that can damage
DNA (60), it may serve as a prototype for a less destructive drug that
kills cells by a mechanism mediated by the MMR pathway. To that end, we
sought to characterize how AD might affect the human MMR pathway
in vitro.
We found that AD, as well as actinomycin D, nogalamycin, and
daunomycin, was capable of inhibiting the correction of G·T or A·C
mispairs by HeLa nuclear extracts when present at low micromolar concentrations. In comparison, AD introduced into cell culture at high
nanomolar concentrations can kill tumor cells. Because AD is both a
potent DNA intercalating agent and relatively hydrophobic, it rapidly
equilibrates into cell nuclei in various tissues within an hour of
subcutaneous injection into hamsters (61). Gigly et al. (62)
have demonstrated that AD-sensitive human K562 leukemia cells in
culture containing 0.1 µM AD rapidly concentrate it to a
nuclear concentration of 11 µM, as measured by
microspectrofluorometry. At other doses of AD, a 100-fold concentration
was also observed. These data are consistent with the hypothesis that
cytotoxic doses of AD reach nuclear concentrations capable of
inhibiting mismatch correction in vivo.
When we found that AD interfered with mismatch correction in
vitro, we initially focused on demonstrating whether the effect could be explained either by reductive activation of molecular oxygen
(60) or by type II topoisomerase inhibition (38). When considered in
the context of the drugs subsequently identified as MMR inhibitors,
such as nogalamycin, actinomycin D, and the weaker inhibitor ethidium
bromide, neither model remains attractive. First, it is unlikely that
the inhibitors are all reduced enzymatically to produce reactive
species, either oxygen or drug-based radicals, that generate DNA damage
capable of inhibiting MMR at similar concentrations. Experimentally, we
found no evidence for covalent DNA modification capable of inhibiting
repair under the assay conditions. AD incubated with either
heteroduplex substrate or HeLa extract alone under mock assay
conditions does not result in MMR inhibition if the AD is removed or
diluted prior to the assay.
If all four compounds acted via a specific type II topoisomerase, then
such inhibition would have to directly or indirectly impact MMR. The
only result consistent with topoisomerase inhibition is the block to
DNA catenation, which occurs concurrently with MMR inhibition. However,
isolation of unrepaired, supercoiled, heteroduplex molecules is also
consistent with ligation of the nicked substrate to a covalently closed
molecule in the presence of an intercalator (63), which is expected to
yield a positively supercoiled molecule following AD removal by phenol
extraction. The inhibition of MMR by AD also stands in contrast to the
nonintercalating or weakly intercalating topoisomerase inhibitors such
as etoposide or m-amsacrine that failed to affect MMR at any
dose tested. A more attractive explanation for the observed MMR
inhibition is that reversible DNA intercalation prevents mismatch
recognition by human MutS In this work, a correlation was observed between the relative
association constants of the tested drugs for DNA intercalation and the
concentrations that inhibited MMR. Unfortunately, no single study has
determined binding affinities for all the drugs studied here under
comparable experimental conditions. This prevents a simple comparison
of drug concentrations that inhibit MMR with their affinity for DNA
intercalation, because the association constants measured are highly
dependent upon the DNA chosen, the experimental conditions, and the
approach used. Perhaps the best insight comes from studies where
association constants were determined for pairs of drugs in the same
study. Consistent with the relative ranking of MMR inhibitors,
daunomycin binds ~1.5-4-fold less strongly than AD (43-45), whereas
ethidium bromide binds DNA roughly 7-fold less tightly than AD (48),
which has a dissociation constant of roughly 300 nM. These
binding data quantitatively recapitulate the drug ranking as MMR
inhibitors, with the effective concentration for MMR inhibition in
crude nuclear extracts roughly 10-fold above the measured dissociation
constant for binding to calf thymus DNA.
A correlation was also observed between DNA binding affinity of the
intercalating drugs and the concentration range over which inhibition
occurs. The strongest DNA binders (AD, nogalamycin, and actinomycin D)
inhibit MMR as the drug concentration varied over 1-5
µM, implicating a highly cooperative step in the
inhibition. Molecules with weaker affinity for DNA, such as ethidium
bromide, show inhibition profiles spread over a broader range. In both cases, the origin of this apparent cooperativity is unclear. Although AD, actinomycin D, and daunorubicin do exhibit positive cooperative binding at low drug occupancies (r < 0.1 mol of drug
bound per mol of base pair at 0.1 M NaCl; Ref. 43), no
evidence for positively cooperative binding at higher concentrations
was observed. Inhibition of MMR in vitro and mismatch
binding occurs at concentrations above the dissociation constant, where
little cooperativity was observed.
Considering both the mutagenic potential of DNA intercalators and their
ability to inhibit MMR in vitro, it is useful to revisit possible mechanisms of mutagenesis. Intercalators such as ethidium bromide have been suggested to decrease the fidelity of DNA replication by increasing the frequency of frameshift mutations and single base
substitutions (64). By disrupting the ability of a replicative polymerase to select or insert the correct base, an increase in biosynthetic errors would result. Such errors must also either escape
surveillance by the MMR pathway or exceed its capacity for mismatch
correction. Based on this work, it is also plausible that a diminution
of MMR efficiency contributes to the reduced replication fidelity in
the presence of DNA intercalators. Specifically, drug intercalation
proximal to a mismatch might prevent its recognition.
The limited data available concerning the effect of AD on replication
fidelity suggest it to be a mutagen in Chinese hamster ovary cells (65,
66). At concentrations that kill roughly 80% of cultured Chinese
hamster ovary cells, a 10-fold increase in mutation frequency at the
xanthine-guanine phosphoribosyltransferase gene locus was determined,
of which 35% of the characterized mutations were large deletions that
removed at least one exon (66). This is not consistent with a complete
loss of MMR, which might be expected to increase the mutation rate from
100- to 1000-fold (67). However, it is not yet possible to characterize
the extent to which a putative interference with MMR contributes to the
number or spectrum of the observed mutational events.
In attempting to dissect the mechanism by which AD reduces replication
fidelity, it must be appreciated that this intercalator shows
pleiotropic effects on DNA metabolic activities. For example, AD has
been shown to inhibit viral DNA polymerases (56, 68) murine DNA
helicase activity (69) and mammalian type II topoisomerase (37).
Adriamycin's best characterized effect, inhibition of mammalian type
II topoisomerase activity, occurs at high nanomolar concentrations
(37). Furthermore, decatenation assays designed to test topoisomerase
catalytic function indicate that in MCF-7 tumors, AD fully inhibits
type II topoisomerase activity at 5 µM (70). At drug
concentrations that inhibit MMR, DNA metabolism in general is likely to
be substantially altered because of inhibition of multiple essential activities.
Another conceivable target for a drug such as AD is an interaction with
one of the MutL homologs essential for mismatch correction, such as
human MLH1 or PMS2. Recently, an N-terminal fragment of the
Escherichia coli MutL protein was shown to have structural similarity to the ATP-binding domain of the DNA gyrase B subunit, an
E. coli type II topoisomerase (71). The gyrase inhibitor novobiocin binds to and inhibits the ATPase function of MutL (71). Although no x-ray crystallographic structure is available for either
subunit of hMutL Our working model to explain how AD inhibits MMR in vitro
does not readily conform to published hypotheses that connect low level
drug resistance with repair loss. The most popular models depend on
damage sensing by MutS Explanations that attempt to reconcile the failure of MutS as if it were an insertion mismatch. Inhibition apparently
results from the ability of the intercalated drug to prevent mismatch
binding, shown using a defined mobility shift assay, which occurs at
drug concentrations that inhibit repair. These data suggest that
adriamycin interacts with the mismatch repair pathway through a
mechanism distinct from the manner by which covalent DNA lesions are processed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(MSH2/MSH6) and MutL
(MLH1/PMS2) at a
lesion, perhaps in concert with other proteins, followed by signaling
cascades that initiate apoptosis. Both models rely on initial damage
recognition by MutS
with a later dependence on other repair and
signaling proteins. Mutations that disrupt MutS
or MutL
activity
may therefore cause drug resistance because this early component of an
apoptotic signaling cascade is absent.
, a heterodimer composed of hMSH2-hMSH6,
(23, 24), appears to be recognition of mispaired bases and small base
insertion mismatches (23, 25, 26). However, band shift experiments
performed with purified MutS
and lesion-containing oligonucleotides
(17, 22) support the hypothesis that specific types of damage are
recognized by MutS
, implicating this activity in the cellular
response to DNA-modifying drugs. The most abundant (27) and likely the
most cytotoxic (27, 28) lesion caused by cisplatin is a 1,2-intrastrand
cross-link between adjacent purines. MutS
binds this adduct (17,
22), as does MSH2 (18). At least one of the cytotoxic modifications
caused by
N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and temezolomide
(O6-methylguanine) is both recognized by MutS
(17) and processed by the MMR pathway (12). Although cisplatin and
alkylators generate a range of DNA lesions, it appears that the
suspected cytotoxic lesions are tightly bound by MutS
(17),
consistent with a requirement for lesion recognition by MutS
for
drug-induced apoptosis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
495 = 13,000 M
1 in methanol (30); daunomycin,
480 = 11,500 M
1 in water (31);
nogalamycin,
258 = 24,755 M
1
in water (Merck Index); ethidium bromide,
480 = 5,600 M
1 in water (Sigma); actinomycin D,
443 = 24,400 M
1 in methanol
(Sigma). Stock solutions were stored for short periods at
20 °C.
3'
from the nick were constructed using published protocols (29, 32). To
prepare A·C heteroduplexes with 10, 128, or 808 bp between the
mispair and nick, the replicative form of f1MR3 was linearized with
XbaI, Sau96I, or HincII, respectively. Strand denaturation, followed by annealing with the appropriate f1MR1
single-strand phage DNA partner, which is complementary except for the
mismatch site, generates the A·C mismatch and places the nick at the
initial restriction site. All subsequent steps in substrate preparation
and purification were as described.
Isolation--
HeLa
nuclear extract was prepared from cells obtained from the National Cell
Culture Center (Minneapolis, MN) according to Ref. 29. MutS
was
purified from HeLa extract as described in Ref. 23.
Drosophila DNA type II topoisomerase was a gift from Dr.
Tao-hsieh Shih (Duke University) and was isolated as described in Ref.
33. Protein concentrations were determined with the Bradford assay
(Bio-Rad) using bovine serum albumin as a standard.
--
Band shift experiments
were performed using protocols described in Ref. 17. Oligonucleotides
of the sequence: 5'-GAATTGCTAGCAAGCTTTCGAGTCTAGAAA-3' (30-mer oligonucleotide 1), a homoduplex complement (30-mer
oligonucleotide 2) and the heteroduplex complement:
5'-TTTCTAGACTCGAGAGCTTGCTAGCAATTC-3' (30-mer oligonucleotide
3) were synthesized by GENOSYS. The site of the mispair is indicated in
bold. Oligonucleotide 1 was labeled with [
-32P]ATP
(Amersham Pharmacia Biotech; 3000 Ci/mmol) using T4 polynucleotide kinase (New England Biolabs). Unincorporated label was removed by size
exclusion chromatography using a 0.75-ml Sephadex G25 micro-spin
column. Homoduplex and heteroduplex oligonucleotides were made by
annealing labeled oligonucleotide 1 with 3 or labeled oligonucleotide 1 with 2, respectively. Oligonucleotides were heated in a solution
containing 10 mM Tris·HCl (pH 7.6), 1 mM EDTA, and 50 mM NaCl for 5 min at 70 °C then allowed to
anneal by incubating for 1 h at 37 °C. The annealed
oligonucleotides were then recovered from a 15% polyacrylamide gel in
1× TAE followed by electroelution into a dialysis membrane. Cold
homoduplex competitor was made using the same protocol except that
oligonucleotide 1 was not radiolabeled.
, and the duplex oligonucleotides. The drugs tested were diluted into the same buffer and added to the
reaction prior to MutS
addition. Once MutS
was introduced into
each experiment by pulse centrifugation, reactions were placed on ice
for 10 min. After incubation, 1.5 µl of load buffer containing 50%
sucrose and 0.05% bromphenol blue in 25 mM Hepes·KOH (pH
7.5) was added to each reaction and then immediately loaded onto a 6%
acrylamide minigel (Bio-Rad). Gels were run at 12 V/cm for 15 min, and
bands were visualized by autoradiography with Kodak X-Omat film and
Kodak MS intensifying screen. These experiments were also replicated in
the presence of 21 fmol of homoduplex F1MR3 closed circular DNA instead
of A·T oligonucleotide competitor to ensure that the base content and
sequence for AD intercalation were the same between repair and
bandshift assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, the heterodimer primarily
responsible for mismatch recognition (23). However, it is less clear
how a drug that reversibly intercalates into DNA might trigger a
MMR-dependent cytotoxic response.
View larger version (33K):
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Fig. 1.
Molecular outcomes when circular 6.4-kb G·T
mismatched substrate (80 ng) is incubated with AD under MMR assay
conditions (a 20-min incubation at 37 °C in the presence of 45 µg of HeLa nuclear extract). Panel
I, the upper arrow identifies linearized, unrepaired
substrate recovered from the in vitro assay, whereas the
lower arrow marks two bands of 3.1 and 3.3 kb that are
diagnostic for strand-specific mismatch correction based on restriction
enzyme sensitivity (see "Materials and Methods"). AD concentrations
are given below in µM, and a graphical representation of
the data is presented in Fig. 3. Panel II, mispaired
substrate was incubated with 1, 8, or 30 µM AD under MMR
assay conditions and then either digested as in panel I to
score for MMR or loaded onto the gel as recovered from the assay (lanes
labeled as undigested). The upper arrow points to catenated
substrate, and the lower arrow points to supercoiled
substrate. Panel III, AD does not cause covalent DNA damage
that affects MMR under the assay conditions in the absence of HeLa
nuclear extract. In the presence of 10 µM AD, MMR
activity is inhibited in vitro (compare lanes 1 and 2). In parallel, the G·T substrate was incubated with
the same concentration of AD under mock assay conditions (omitting only
HeLa nuclear extract; see "Materials and Methods") and then
recovered by ethanol precipitation. This treatment has no effect on the
ability of the AD-treated substrate to participate in a subsequent
assay for MMR in vitro (compare lanes 3 and
4).
may recognize intercalated AD as a frameshift
mispair. This could titrate MutS
away from binding legitimate
mispairings, represented by G·T or A·C in the case of the in
vitro MMR assay. 2) AD could prevent mispair binding, either by
interacting directly with MutS
or by intercalation proximal to the
mismatch site. 3) AD could cause covalent DNA damage that is capable of
sequestering MMR components away from the G·T mispair. 4)
Intercalated AD molecules could physically hinder the movement of
proteins required for early steps in MMR, such as MutS
, along the
DNA contour. 5) AD is known to inhibit type II topoisomerase activity
(37), which could directly or indirectly impact MMR.
View larger version (23K):
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Fig. 2.
Early steps in human mismatch repair could be
inhibited by several possible mechanisms. A mispair in DNA (*) is
normally bound by MutS (MSH2/MSH6), which is followed by an
interaction or reaction with ATP that leads to movement along the DNA
helix. Not shown is MutL
(MLH1/PMS2), which also plays an
essential role at an early stage. The hypotheses we tested to explain
how AD might inhibit MMR are presented on the right side of
the panel. Hypothesis 1, adriamycin (black rectangle) could
block repair because it is recognized as a base insertion mismatch and
thereby titrate MutS
away from the G·T mismatch. Hypothesis
2, mismatch binding could be hindered in the presence of AD,
perhaps by a physical distortion of the DNA helix. Hypothesis
3, oxidative damage might be formed that either blocks mispair
processing or is recognized as a mismatch. Hypothesis 4,
MutS
movement along the helix might be physically impeded by
intercalated AD. Hypothesis 5, AD inhibition of a type II
topoisomerase might indirectly inhibit MMR by altering the topological
or catenation state of the substrate molecule undergoing repair.
View larger version (14K):
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Fig. 3.
Mismatch correction is inhibited by DNA
intercalators. Mismatch repair values are normalized relative to
the percentage of repair in the absence of drug. A relative value of 1 represents 8-10 fmol of correction of G·T to AT in the absence of
drug. Top panel, actinomycin D ( ) and nogalamyin (×)
inhibit mismatch repair at concentrations effectively indistinguishable
from AD (
), whereas daunomycin (
) is a 2-fold less effective
inhibitor. Bottom panel, ethidium bromide blocks repair at a
10-fold higher concentration. A linear curve fit was arbitrarily
applied to the binding data.
1) compared with
AD (KA = 3.6 × 106
M
1, taken from Ref. 48). These data indicate
that the characterized inhibition of type II topoisomerase activity by
AD is likely to be distinct from its ability to inhibit MMR, because
other topo II inhibitors do not inhibit MMR. However, in the case of
etoposide or m-amsacrine, a block to substrate catenation
was not observed in vitro, as it was in the case of AD (Fig.
1).
, along the DNA
helix, we constructed three molecules with varying distances separating a single-strand nick and an A·C mispair. The nick serves to direct MMR to the discontinuous strand in human MMR assays (29, 54). Furthermore, Fang and Modrich (55) have demonstrated that a tract of
DNA that spans the mismatch and nick is excised during mismatch
correction. Mispaired substrates containing nicks positioned 10, 128, or 808 bases away from an A·C mismatch were assayed for repair in the
presence or absence of AD. When the nick is placed 10 bases away from
the mispair, there are few potential AD intercalation sites between the
mispair and nick, especially considering that AD intercalation
saturates, on average, at once every 5-6 bp (44, 56). The number of
potential intercalation sites is increased over 130-fold in the
substrate with an 808-bp separation. Assuming that the base content
across this span of DNA is not highly skewed against intercalation,
more physical "roadblocks" will be present as the distance
increases at a given drug concentration.
View larger version (15K):
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Fig. 4.
The block to mismatch repair is independent
of the distance between the mispair and nick. The relative extent
of repair was determined in the presence of 0-5 µM AD
for three G·T substrates where the mismatch and repair-directing nick
are separated by distances of either 10 bp ( ), 128 bp (×), or 808 bp (+). These relative distances are drawn on a representative
substrate molecule above the plot to illustrate the variable number of
potential AD binding sites between the mispair and nick. The
restriction enzymes used to create each nick are also given (see
"Materials and Methods").
(MSH2/MSH6; Ref. 23). When the nick and
mismatch are separated by 10 bp, a repair event with similar
characteristics and magnitude is obtained where at least 75% of the
nick-directed mismatch correction is dependent upon MSH2 (data not
shown). The fraction of repair that is MSH2-independent (up to 25%,
depending upon the substrate and extract) may represent exonucleolytic
activity from the extract initiated at the nearby nick or an
alternative repair pathway.
footprint.
--
It
has been shown that MutS
can recognize 1,2-platinated adducts formed
between cisplatin and DNA, as well as specific alkylation damage such
as O6-methylguanine (17, 22). Upon intercalation
into DNA, AD might appear as a base insertion mismatch, and this adduct
might be recognized by MutS
. A plausible explanation for inhibition
of MMR in vitro might be that, in the presence of
intercalated AD, the limited supply of MutS
is titrated away from
the G·T mismatch where repair is monitored. To test this possibility,
we performed band shift assays with MutS
and 30-bp A·T homoduplex
and G·T heteroduplex oligonucleotides in the presence of increasing
concentrations of AD (Fig. 5). These
oligonucleotides are identical in sequence to a 30-bp region
surrounding the G·T mismatch in the assay substrate. In these
experiments, homoduplex and heteroduplex control oligonucleotides were
incubated with AD prior to the addition of 100 ng of MutS
. If DNA
containing intercalated AD is recognized as a mispair, a ternary
complex between MutS
, AD, and oligonucleotide is expected with a
mobility in native gels similar to the complex between MutS
and
G·T heteroduplex oligonucleotide.
View larger version (23K):
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Fig. 5.
DNA-intercalated AD is not bound by
MutS . 4 fmol of A·T homoduplex or G·T
heteroduplex oligo, plus 200 fmol of homoduplex competitor, were
preincubated with AD at the concentrations shown (in µM)
before 100 ng of MutS
was added. No protein was included in the lane
marked "
". The arrow indicates the complex between
MutS
and heteroduplex oligonucleotides, which does not form in the
presence of higher AD concentrations.
was presented with
oligonucleotides that contained, on average, a range of less than one
to several intercalated AD molecules. Incubations were performed in the
presence of either unlabeled homoduplex competitor oligo or 21 fmol of f1MR3 viral double-stranded DNA to parallel the DNA base content and
stoichiometry present in the MMR assay. As shown in Fig. 5, no evidence
for a ternary complex involving MutS
, AT homoduplex, and AD was
observed at any drug concentration.
bound G·T heteroduplex oligonucleotides in the
absence of AD (Fig. 5). However, when AD was present at 3.3 or 10 µM, a sharp decrease in the ability of MutS
to form a
stable complex with the mismatch was observed. These results were
independent of the order in which the reagents were assembled; if
MutS
was added to the reaction first, to allow binding with the
mismatch, and AD added last, the same results were obtained (data not
shown). This suggests that AD can disrupt mispair binding by MutS
,
even if MutS
is already bound to the heteroduplex. In parallel with
inhibition of MMR activity, the block to mispair binding by MutS
in vitro was observed over the same narrow concentration range where AD also blocks the MMR assay (compare Fig. 1 with Fig.
5).
to G·T heteroduplexes in the
presence of the drug (Fig. 6; data not
shown for ethidium bromide) was disrupted over a concentration range
similar to that which inhibited MMR in vitro (compare Figs.
3 and 6). As with AD, this bandshift disruption occurred whether
MutS
or drug was added first (data not shown). From these data, it
appears likely that repair of the G·T substrate is impeded because
MutS
does not recognize mispaired DNA in the presence of the
intercalating drug. The parallel with AD is not exact, however, because
AD completely blocked complex formation between MutS
and the
mismatch at 10 µM, whereas a fraction of the bound
complex remains in the presence of actinomycin D or nogalamycin at 10 µM (Fig. 6). MMR was not detectable above concentrations
of 5 µM in the presence of any of the three drugs.
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Fig. 6.
Nogalamycin and actinomycin D block
MutS binding to mispaired
oligonucleotides. Band shift experiments with G·T heteroduplex
oligonucleotide were done as in Fig. 5. The first lane
contains only radiolabeled oligonucleotide (4 fmol, lower
band). All other lanes have 4 fmol G·T heteroduplex and 100 ng
of MutS
plus the indicated concentration of drug. The top
band is the bound complex between MutS
and heteroduplex
oligonucleotides.
and the G·T mispair.
Specifically, we cannot distinguish between an inhibition based on drug
intercalation and a direct interaction of the drug with either MutS
or the MutS
/DNA complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
.
(MLH1/PMS2), it is plausible that the highly
conserved ATP binding domain represents a similar structure. Because AD
can inhibit eukaryotic topoisomerase activity, it is seductive to
consider the possibility that its target for MMR inhibition is a
catalytic function contributed by human MutL
. This possibility seems
less likely because it would implicate a highly similar binding
constant for MutL
shared by structurally distinct intercalating
compounds. The strong correspondence between DNA binding affinity and
loss of both mismatch recognition and repair does not rule out such an
effect, and this possibility has not been tested experimentally.
, which is proposed to initiate a series of
events resulting in apoptosis. Consistent with this hypothesis,
adjacent platinated guanines (17, 22) and
O6-methylguanine (17) are recognized by MutS
in mobility shift assays, and O6-methylguanine
adducts are removed by the MMR pathway in vitro when strand
breaks are placed in the same strand as the lesion (12). In contrast,
MutS
does not form complexes with oligonucleotides incubated with AD
over concentration ranges that offer one to several "lesions" per
oligo. Instead, drug intercalation apparently disrupts structural
features in DNA used by MutS
to distinguish mismatch-containing DNA
within correctly base paired flanking DNA. So, although lesion
recognition by MutS
is the implied prerequisite for initiating a
damage signal cascade, we can find no evidence for a ternary complex
between MutS
, AD, and DNA.
to bind
intercalated AD with the futile repair or direct signaling models are
speculative. A formal possibility is that intercalated AD may in fact
be recognized as a mismatch but in a specific DNA sequence context not
found in our substrate molecule. It is still plausible that the
presence of AD leads to chromosomal DNA damage or specific oxidized
lesions. Such secondary rearrangements or covalent lesions could be
recognized by MutS
and initiate a MMR-dependent apoptotic cascade. One could also imagine distinct but related mechanisms; cisplatin and DNA alkylators might kill cells by triggering lesion processing subsequent to recognition by MutS
, whereas AD
initiates cell death through a distinct MMR-dependent
mechanism. For example, the activity that initiates processing of
intercalated bases might be MutS
(MSH2/MSH3) rather than MutS
.
Alternatively, one could imagine that AD, cisplatin, and alkylating
drugs all trigger same MMR-dependent mechanism, but neither
the futile repair nor direct signaling models are the correct
explanations for drug resistance. Recently, Brown et al.
(34) have suggested that covalent adducts such as cisplatin lead to
MLH1-dependent replication stalling, and AD intercalation
might also stall replication independent of its ability to inhibit MMR.
It is therefore conceivable that the inhibition of MMR we used to
demonstrate an interaction with AD is not an appropriate model system
to understand the connection between the loss of MMR function and the
low level resistance to AD.
![]() |
ACKNOWLEDGEMENTS |
---|
We recognize the excellent cell culture service provided by the National Cell Culture Center (Minneapolis, MN). Dr. Keith Iams and Dr. Martina Celerin also provided thoughtful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Genetics, Cellular, and Molecular Sciences Training Grant GM 07757-21 (to E. D. L.) and by funds from the American Cancer Society and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
812-856-4184; E-mail: jdrummon@bio.indiana.edu.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M006390200
2 J. T. Drummond, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MMR, DNA mismatch repair; AD, adriamycin; bp, base pair(s); kb, kilobase(s).
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