Download The Role of DNA Mismatch Repair in Platinum

(CANCER RESEARCH56, 4881-4886, November 1. 19961
Advances in Brief
The Role of DNA Mismatch Repair in Platinum Drug Resistance1
Daniel Fink,2 Sibylle Nebel, Stefan Aebi, Hua Zheng, Bruno Cenm, Alissar Nehmé,Randolph
D. Christen,
and Stephen B. Howell
Department of Medicine and the Cancer Center, University of California at San Diego, La Jolla. California 92093-i'JWJS8
Abstract
Loss of DNA mismatch repair occurs in many types of tumors. The
effect ofthe loss ofDNA mismatch repair activity on sensitivity to cisplatin
and a panel of analogues was tested using two pairs of cell lines proficient
or deficient In this function. HCT116+ch2, a human colon cancer cell line
deficient In hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resist
ant to carboplatin when compared to a subline complemented with chro
mosome
3 expressing
endometrial
cancer
a wild-type
copy
cell line HECS9,
of hMLH1.
which
Likewise,
Is deficient
the human
in hMSH2,
was
1.8-foldresistantto cisplatinand 1.5-foldresistantto carboplatinwhen
compared
to a sublime complemented
with chromosome
2 with a wild-type
hMSH2. In contrast to cisplatin and carboplatin, which form the same
types ofadducts in DNA, there was no difference In sensitivity between the
DNA mismatch repair-proficient and -deficient cell lines for oxaliplatin,
tetraplatin,
transplatin,
JM335,
or JM216.
The formation
of protein-DNA
complexes that contained hMSH2 and hMLH1 was documented by mo
bility shift assay when nuclear extracts were incubated with DNA plati
nated with cisplatin but not with oxaliplatin. These results demonstrate a
correlation
between
failure
of the
DNA
mismatch
repair
proteins
to
recognize the platinum adduct and low-level resistance, suggesting a role
for the DNA mismatch repair system in generating signals that contribute
to the generation of apoptotic activity. They also identify the use of drugs
whose
adducts
are not recognized
as a strategy
for circumventing
resist
ance due to loss of DNAmismatchrepair.
Introduction
Cisplatin [cis-diamminedichloroplatinum]
and carboplatin [cis-di
ammine(1,1-cyclobutanedicarboxylato)platinum(II)]
are chemothera
peutic agents in widespread use for the treatment of a variety of
human malignancies. However, many tumors are intrinsically resist
ant to these drugs, and the development of acquired resistance during
the course of treatment of even initially sensitive tumors is a common
occurrence that constitutes a major obstacle to the curative use of
these drugs. For example, the majority of human ovarian carcinomas
respond initially to treatment with these agents, but most of these
tumors recur, and when they do, most are resistant to treatment with
both cisplatin and carboplatin (1). Changes of <2-fold in sensitivity to
cisplatin are sufficient to account for treatment failure in human tumor
xenografts (2). Resistance to cisplatin and carboplatin in cell lines
selected with these agents in vitro is multifactorial (reviewed in Ref.
3), but partially because low levels of resistance are sufficient to cause
Received 8/12/96; accepted 9/18/96.
The costs of publication of this article were defrayed in part by the payment of page
charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith
18 U.S.C. Section 1734 solely to indicate this fact.
I Supported
in part by Grant CTR4154
from the Council
for Tobacco
Research,
grants
lack of clinical responsiveness, it has been difficult to identify those
mechanisms that are of the greatest clinical importance.
We have recently reported the novel finding that loss of DNA
mismatch repair produces low-level resistance to cisplatin (4). Loss of
DNA mismatch repair is the predisposing factor in hereditary non
polyposis colon cancer and occurs in a wide variety of sporadic
human cancers as well (reviewed in Ref. 5). Loss of mismatch repair
has been reported to cause moderate levels of resistance to the
methylating agent MNNG3 (6) and high-level resistance to the anti
metabolite 6-thioguanine (7). The fact that the mismatch repair system
proteins can recognize the DNA distortion produced by the presence
of 6-thioguanine and the adducts produced by both MNNG and
cisplatin (8) suggests that the DNA mismatch repair proteins serve to
detect the DNA damage caused by these agents and generate an injury
signal that eventually contributes to the triggering of the apoptotic
reaction that destroys the cell.
No information is currently available on whether loss of DNA
mismatch repair causes resistance to other platinum-containing drugs
in addition to cisplatin. We have investigated the effect of the loss of
mismatch repair on sensitivity to a variety of cisplatin analogues that
produce either identical, similar, or quite different adducts in DNA
and report here that resistance due to the loss of mismatch repair is
specific to cisplatin and carboplatin. Failure of the loss of mismatch
repair to cause resistance to platinum-containing analogues containing
a diaminocyclohexane group is linked to failure of the DNA mismatch
repair proteins to recognize this type of adduct and form complexes on
platinated DNA probes. Of particular importance is the observation
that loss of mismatch repair does not cause resistance to oxaliplatin or
JM216, two new platinum analogues now in clinical evaluation
(9, 10).
Materials and Methods
Cell Lines. The humanovarianadenocarcinomacell line 2008 (11) was
maintained
as were
nologies,
hMLH1
akademischen
Nachwuchses
zur Forderung
des
1640 supplemented
with
the hMSH2-deficient
human
endometrial
adenocarcinoma
cell line
HEC59 (13) and a subline complemented with chromosome 2 (clone HEC59/
2-4). Both cell lines were maintained in Iscove's modified Dulbecco's medium
(Irvine Scientific, Irvine, CA) supplemented with 2 m@vt
L-glutamineand 10%
heat-inactivated fetal bovine serum. All the chromosome-complemented lines
were maintained in medium supplemented with geneticine (400 @xg/ml
for
HCT1l6+ch2 and HCI'l l6+ch3 and 600 @g/ml
for HECS9+ch2; Life Tech
of Cancer ofthe
Award from the Kommission
at 37°Cin RPMI
deficient human colorectal adenocarcinoma cell line HCT116 was obtained
from the American Type Culture Collection (ATCC CCL 247); sublines
complemented with chromosome 3 (clone HCT116/3-6) and chromosome 2
(clone HCT116/2-1) were obtained from Drs. C. R. Boland and M. Koi (12),
from the American Society of Clinical Oncology and from the Association for the Cure
Prostate, by a Fellowship
in a 5% CO2 atmosphere
2 m@iL-glutamine and 10% heat-inactivated fetal bovine serum. The hMLH1-
Inc., Gaithersburg,
in HCT1I6+ch2
MD). The absence
and HCTI16+ch3
and presence
cells
of expression
of
as well as expression
of
of the University of Zurich to D. F., and by a Fellowship
Award from the Ernst ScheringResearchFoundation,Berlin, and the EMDO Stiftung,
Zurich, to S. N. This work was conducted
in part by the Clayton Foundation
for
Research-California
Division.R.D.C. andS. B.H.areClaytonFoundationInvestigators.
D. F. and S. N. contributed equally to this work.
2 To
whom
requests
for
reprints
should
be
addressed,
at
the
Department
of
3 The
abbreviations
used
are:
MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
JM2I6,
bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV);
JM335,
trans-ammine
(cyclohexylaminedichlorodihydroxo)platinum(IV);
oxaliplatin, [trans-(L)-l,2-diaminocy
clohexane]oxalatoplatinum(II);
tetraplatin,(d,l)trans-l,2-diaminocyclohexanetetrachlo
Medicine
0058, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093.
Phone:(619)822-1110;Fax:(619)822-1111.
roplatinum(IV); poly(dI@dc),poly(deoxyinosinic-deoxycytidylic acid); GTBP, GIF bind
ing protein.
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DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE
hMSH2 in HEC59 and HEC59+2 cells were verified by immunoblot analysis
(data not shown). All cell lines tested negative for contamination with Myco
plasma
HN
spp.
@
transplatin
Centre
were purchased
(Reading,
from Sigma
United
Chemical
Kingdom).
Tetraplatin
Co. (St. Louis,
,oc
Pt
HN
sanne, Switzerland). JM216 and JM335 were kindly provided by the Johnson
Technology
HN\
0
3\/
Materials. Cisplatin and carboplatin were obtained from Bristol-Myers
Squibb Co. (Princeton, NJ). Oxaliplatin was a gift from Debiopharm (Lau
Matthey
ci
/
Pt
/\
ci
HN
o—c
3
and
Cisplafin
MO).
Carboplatin
Cytotoxicity Assays. Cisplatin, carboplatin, oxaliplatin, tetraplatin, and
JM335weredissolvedimmediatelybeforeusein 0.9%NaCIsolutionat 1mz@i,
@
whereas stock solutions of JM216 and transplatin were prepared in DMSO.
The final concentration of DMSO in the cultures was <0.1% at all drug
concentrations and in controls. Clonogenic assays were performed by seeding
250 cells from a single-cell suspension into 60-mm plastic dishes in 5 ml of
media. After 24 h, appropriate amounts of the drugs were added to the dishes,
and the cells were exposed for 1 h. Thereafter, the cells were washed, and new
drug-free medium was added. Colonies of at least 50 cells were scored visually
after 8—10
days for HCT116 and 12—14
days for HEC59. Each experiment was
performed a minimum of three times using triplicate cultures for each drug
concentration.
IC,0 values were estimated using logarithmic interpolation at a
relative plating efficiency of 0.5.
DNA Fragment Preparation. A l23-bp DNA ladder(Life Technologies,
Inc.) consisting of various numbers of a single l23-bp fragment ligated in
various
copy
numbers
head-to-tail
was platinated
overnight
by incubation
NH
/
end-labeled
using [y-32P]ATP
(3000 Ci/mmol;
DuPont
New England
/F@\
NH@1 ci
:@
Oxaliplatin
Tetraplatin
KII:@—
Pt \/ 3
ci
OH
3
/I\
NH
I
2
NH
\/
Nuclear,
molar ratio of 0.05 served as a competitor to identify specific
binding. A Perkin Elmer Cetus model 373 atomic absorption spectrophotom
eter was used to quantitate the extent of platination after exposure to cisplatin
or oxaliplatin.
JM335
HN@a 0
/f____\\,/
ci
NH
Pt
ci
OH
HN
3
at
Boston, MA) and T4 polynucleotide kinase (Promega, Madison, WI). Unin
corporated label was removed by chromatography on Sephadex G50 (Phar
macia Biotech Inc., Piscataway, NJ). Salmon sperm DNA platinated at a
drug:nucleotide
N
I
\Io—c
NH
37°C
with aquated cisplatin or aquated oxaliplatin diluted in 10 mMNaCIO4at
a drug:nucleotide molar ratio of 0.05. The platinated DNA ladder was precip
itated in ethanol and digested to completion with AvaI (Life Technologies,
Inc.) to reduce all fragments to 123 bp. This fragment contains 13 0(1 pairs
that are potential sites for intrastrand d(GpG) cross-links and 11 potential sites
for d(ApG) cross-links. The digest was analyzed by electrophoresis in 2%
agarose. The 123-bp DNA fragment was recovered from the gel, cleaned, and
ethanol-precipitated. DNA quantitated by spectrophotometry at 260 nm was
@
0—c
0
ci
Transpiatin
cH 3
NH2
\@__/
@0
JM21Ô
Fig. I. Structures of the platinum-containing
drugs studied.
Nudear Extract Preparation. Cells were harvestedduring exponential
growth, washed in buffer [10 msi HEPES (pH 7.4), 10 mMKC1,and 0.5 mM
DTT], and lysed at 4°Cin the same buffer containing 1% NP4O.The nuclei
were collected by centrifugation and lysed in high-salt buffer [20 mMHEPES
(pH 7.9), 500 mMNaC1,0.2 mMEDTA, 0.5 mi@iphenylmethylsulfonyl fluo
ride, 0.5 mMDTF, 1.5 mr@i
MgCI2,and 20% glycerol] at 4°C.The supernatant
was designated the nuclear extract and stored at —70°C
(14). Protein concen
trationsweredeterminedby the methodof Bradford.
Pt(II), and the chlorides and moieties attached to the platinum atom
via oxygen atom linkages are displaced by water to produce aquated
forms of the drug. The moieties attached via the nitrogen atoms
remain associated with the platinum atom even after it has formed an
adduct with DNA. Thus, after aquation, cisplatin and carboplatin
produce the same types of adducts in DNA as do oxaliplatin and
MObIlity Shift Assays. The platinated and radiolabeled l23-bp DNA frag
tetraplatin, whereas the structure of the adducts produced by trans
ment (1.5 ng) was incubated on ice for 30 mm with nuclear extract (5 @.&g)
in
platin, JM335, and JM216 are somewhat different. The effect of the
the presence of 2.5 ,.&gpoly(dI-dc)poly(dI@dc)(Sigma) in a final volume of 10
loss of DNA mismatch repair activity on sensitivity to cisplatin and
@d.hMLH1 was detected by supershift using the rabbit polyclonal antibody
these
analogues was tested using two pairs of cell lines. One member
PC56 (Calbiochem, San Diego, CA), and hMSH2 was detected using the rabbit
of each pair was proficient with respect to DNA mismatch repair,
polyclonal antibody PC57 (Calbiochem).
The rabbit polyclonal anti-Bcl-xL PC
whereas the other was deficient; lack of DNA mismatch repair in one
67 (Calbiochem) and rabbit anti-actin (A-2066; Sigma) were chosen as nega
tive controls. Antibody (0.05 @xg)
was added to the incubation mixtures. All of the deficient lines was due to lack of hMSH2 function, whereas in
incubation mixtures were diluted in shift buffer [12 mMHEPES (pH 7.9), 12% the other deficient line, it was due to lack of hMLH1 function.
glycerol, 4 mMTris (pH 7.9), 5 ms@MgCI2, 100 nmi KCI, 0.6 inst EDTA, and Parental HCT1 16 human colon carcinoma cells are DNA mismatch
0.6 msi DTF], electrophoresed with 2 pi of 0.06% bromphenol blue in a 5% repair-deficient due to a deletion of one hMLHJ allele and mutation of
polyacrylamide
gel (29:1, acrylamidthisacrylamide),
dried onto Whatman
the other (13). In the HCTI 16+ch3 subline, the hMLH1 deficiency
3-MM paper, and autoradiographed at —70°C.
was complemented by transfer into the cell of a complete chromo
some 3 containing a wild-type copy of hMLHJ; the HCT1 l6+ch2
Results
subline into which chromosome 2 had been transferred served as a
control (12). Parental HEC59 human endometrial carcinoma cells are
Cytotoxicity Studies. Fig. 1 shows the structures of cisplatin and
the various analogues included in this study. They differ with respect DNA mismatch repair-deficient due to two different mutations in each
allele of hMSH2 (13). In the HEC59+ch2 subline, the hMSH2 defi
to the leaving groups that are displaced as the drug becomes aquated
ciency was complemented by transfer of a full-length chromosome 2
and to the structure of the adducts produced in DNA. In the intracel
lular compartment, the axial ligands are lost as Pt(IV) is reduced to containing a wild-type copy of the gene.
4882
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DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE
Cisplatin
100
100
0
0
>
>
2:
... \@
@10
C
10
ci,
0
ci@
0@
0
5
10
15
20
1
25
0
5
10
1@5 20
25
Oxaliplatin
100
Fig. 2. Clonogenic
survival curves for cisplatin, oxaliplatin,
and
JM216for the mismatch-deficient
(& HCT116+ch2)and -proficient
(0 , HCT1I6+ch3)coloncarcinomacell linesandthe mismatch-defi
cient (A, HEC59) and -proficient (•, HEC59+ch2) endometrial carci
norm cell lines. Each point represents the mean of three to five exper
imentsperformedwithtriplicatecultures.Bars,SD.
0
>
2:
:,
2:
:@
C,)
U)
C
C
a)
0
a)
ci)
0
ci)
0@
0@
10
10
jiM
JM21Ô
100
100
N
0
0
>
>
2:
:@
2:
:@
U)
C/)
C
ci)
C
ci)
0
0
ci)
‘I)
0@
0
20
Fig. 2 shows the survival of clonogenic cells as a function of drug
concentration for three representative drugs (cisplatin, oxaliplatin, and
JM216) for both pairs ofcell lines. The IC50 values for all ofthe drugs
tested are presented in Table 1. The hMLH1-deficient HCT116+ch2
cells were 2.1-fold more resistant to cisplatin than the DNA mismatch
repair-proficient HCT1 16+ch3 cells (IC50, 23.2 ± 3.7 versus
Table 1 IC50 values for the platinum-containing drugs used in DNA mismatch repair
deficient (HCTII6+ch2 and HECS9) and -proficient cells (HCTII6+ch3
and HEC59+ch2)a
HCTI16+ch2
(@&M)Cisplatin
Carboplazin
Oxaliplatin
Tetraplatin
JM335
Transplatin
1.9a
JM2I623.2
Values
arethe
(MM)HCTI16+ch3
(g.CM)HEC59
±3.7
125.2 ±12.0
±3.5
97.9 ±6.1
14.1 ±3.6
30.1 ±8.8
23.3 ±2.2
16.3 ±4.3
33.7 ±10.7
23.3 ±2.0
(@&M)HEC59+ch2
±1.0
51.6 ±3.3
14.9 ±2.2
±1.2
35.0 ±4.3
21.5 ±4.4
15.8 ±1.6
26.0 ±1.7
17.5 ±2.5
17.9 ±2.6
263.4 ±74.0
287.4 ±68.2
221.6 ±38.8
298.3 ±55.1
53.2 ±5.511.2 50.2 ±3.114.0 38.8 ±3.27.9 39.9 ±
mean ±SD of at least three independent experiments in triplicate.
40
60
80
100
10
20
40 60
jiM
80 100
11.2 ±3.5 @LM
SD; n
5; P < 0.05 in a two-sided t test). Likewise,
the DNA mismatch repair-deficient HEC59 cells were 1.8-fold more
resistant to cisplatin than the proficient HEC59+ch2 cells (IC50,
14.0 ±1.0 versus 7.9 ±1.2 @tM
SD; n = 3; P < 0.05 in a two-sided
t test).
Carboplatin
contains
a 1,1-cyclobutanedicarboxylato-leaving
group and undergoes aquation more slowly, but the structures of the
aquated forms of cisplatin and carboplatin are the same as are the
types of adducts produced in DNA. Consistent with this, carboplatin
was 5.4-fold less potent than cisplatin against the HCT116+ch2 cells
and 3.7-fold less potent against the HEC59 cells. However, as for
cisplatin, both DNA mismatch repair-deficient cells were resistant to
carboplatin. The HCT116+ch2 cells were 1.3-fold resistant relative to
the HCT1 16+ch3 cells (IC50, 125.2 ±12.0 versus 97.9 ±6.1 @M
SD;
n = 3; P < 0.05 in a two-sided t test), and the HEC59 cells were
1.5-fold resistant relative to the HEC59+ch2 cells (IC50, 5 1.6 ±3.3
versus 35.0 ±4.3 @.LM
SD; n
3; P < 0.05 in a two-sided t test).
Although the potency of the other drugs tested differed as a func
lion of chemical structure, the data presented in Table 1 show that
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DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE
there was no difference in sensitivity between the DNA mismatch
repair-proficient and -deficient cells for oxaliplatin, tetraplatin, trans
platin, JM335, or JM2I6 over the first two logs of cell kill. This
indicates that, among the drugs tested, platinum drug resistance asso
ciated with loss of DNA mismatch repair activity is specific for
cisplatin and carboplatin and suggests that this is due to differences in
the ability of the DNA mismatch repair proteins to recognize the
different types of adducts produced.
The difference in sensitivity of the mismatch repair-deficient
HCT116 and HEC59 cells was not due to a decrease in the uptake of
cisplatin into these cells or the extent of DNA platination (data not
shown). The l.h cellular accumulation was 303 ±58 fmol4tg protein
(SD) for the HCTI l6+ch2 cells and 289 ±82 fmol/p@gprotein (SD)
for the HCT116+ch3 cells (n = 4; P = 0.80 in a two-sided t test).
Mobility Shift Assays. The ability of mismatch repair proteins to
recognize different types of platinum adducts was examined using
mobility shift assays. Nuclei from the human ovarian adenocarcinoma
cell line 2008 were used as a source of nuclear extract because these
cells express hMLH1 and hMSH2 in amounts that are readily detect
able by immunoblot and had been previously characterized with
respect to their ability to form complexes with platinated DNA (15).
The formation of protein complexes on platinated DNA was detected
by gel mobility shift assay using a l23-bp double-stranded oligonu
cleotide platinated with either cisplatin or oxaliplatin at a drug:
nucleotide molar ratio of 0.05. Fig. 3A shows that, in the absence of
nuclear extract, the l23-bp probe migrated to the bottom of the gel.
When incubated with nuclear extract in the presence of carrier DNA
in the form of poly(dldc)poly(dldc),
three complexes were detected
by virtue of their retarded gel mobility (Fig. 3A, Lane 4). Addition of
a 1000-fold excess of salmon sperm DNA platinated with cisplatin at
a drug:nucleotide molar ratio of 0.05 eliminated the formation of all
three complexes (Lane 3), and incubation of nonplatinated probe with
nuclear extract did not result in the formation of any complexes (data
not shown), both of which establish that complex formation was
specific for platinated DNA. The presence of hMSH2 and hMLH1
proteins in all three of the complexes formed on DNA treated with
cisplatin was documented by the fact that addition of either anti
hMLH1 antibody (Lane 5) or anti-hMSH2 antibody (Lane 6) caused
a supershift of each of the complexes that was not observed with a
control antibody directed against Bcl-xL (Lane 8).
Fig. 3B shows the results of the same type of experiment performed
under identical conditions and run on the same gel with the 123-bp
probe platinated with oxaliplatin. In the presence of carrier
poly(dFdc)poly(dldc)
DNA, the probe formed two complexes (Fig.
3B, Lane 4), and complex formation was eliminated by addition of a
1000-fold excess of salmon sperm DNA platinated with oxaliplatin
(Lane 3). However, neither the anti-hMSH2 (Lane 6) nor the anti
hMLH1 antibody (Lane 5) detected the presence of hMSH2 or
hMLHI in these complexes. This result indicates that different types
of complexes are formed depending on the type of N-containing
adduct in the DNA, and that the DNA mismatch repair complex that
forms on cisplatin adducts does not form on oxaliplatin adducts. This
specificity parallels the fact that loss of DNA mismatch repair func
tion resulted in resistance for cisplatin but not for oxaliplatin, sug
gesting that resistance is related to a failure of the repair system to
recognize the presence of the adduct or generate a signal that can
trigger apoptosis.
DNA, but is not associated with resistance to analogues that produce
several other types of adducts. This observation is important for
several reasons: (a) it identifies a novel molecular mechanism that can
cause cisplatin and carboplatin resistance; (b) it indicates a role for at
least the hMSH2 and hMLH1 components of the DNA mismatch
repair system in the recognition of DNA damage or the generation of
signals that leads to apoptosis; (c) it documents that the components
of the DNA mismatch repair system responsible for the difference in
sensitivity are quite specific in their ability to discriminate between
different types of closely related adducts; and (d) it identifies a series
ofplatinum-containing drugs that might be of therapeutic value for the
treatment of tumors resistant to cisplatin and carboplatin by virtue of
the loss of DNA mismatch repair.
MSH2, either by itself (16) or in conjunction with GTBP/pl6O (17,
18), recognizes small DNA mismatches, and Duckett et a!. demon
strated that hMutSa activity, consisting of a heterodimer of hMSH2
and GTBP/pl6O, can bind to DNA platinated by cisplatin (8), sug
gesting that at least some types of cisplatin adducts are recognized as
the functional equivalent of a nucleotide mismatch by the DNA
mismatch repair system. The results presented in this study extend
these observations by documenting that whether or not a cell can
recognize the adduct via the mismatch repair system has a functional
consequence, in this case, manifest as a change in drug sensitivity.
A priori one might have expected that loss of a DNA repair system
would result in hypersensitivity to cisplatin. There is good evidence
that cisplatin adducts can be removed from DNA by nucleotide
excision repair (19, 20), and cells defective in nucleotide excision
repair are in fact markedly hypersensitive to this drug (21, 22).
However, in the case of the DNA mismatch repair system, loss of
function results in cisplatin resistance. This is true also for two other
drugs that produce distortions in DNA that are recognized by the
DNA mismatch repair complex of proteins 6-thioguanine and MNNG
(8). The current paradigm is that the DNA mismatch repair complex
recognizes the adduct in the template strand and attempts repair of the
newly synthesized nonadducted strand. As long as the adduct persists,
insertion of new bases in the nonadducted strand fails to resolve the
apparent mismatch, and the futile cycling of the attempted repair
results in the generation of a signal that normally causes apoptosis (8).
When the DNA mismatch repair system is defective, presumably this
signal is not generated, and the cell seems to be resistant to the drug.
The structural basis for the ability of hMSH2 to recognize mis
matches is not well defined beyond the fact that the double helix is
distorted. The adducts produced by cisplatin and carboplatin also
distort DNA, and the data are consistent with the concept that it is this
distortion that is being recognized. Information is not currently avail
able on whether hMSH2 or the hMSH2/GTBP complex can distin
guish between the different types of cisplatin adducts, which include
G-G, A-G, G-X-G intrastrand cross-links, G-G interstrand cross-links,
and G monoadducts, although it has been demonstrated that the
hMutSa activity can distinguish between the cisplatin l,2-intrastrand
and the transplatin 1,3-intrastrand adducts (8). The failure to recog
nize the adducts produced by oxaliplatin may be a result of a differ
ence in the distortion produced by its adducts in DNA, or it could be
due to steric hindrance of the binding of hMSH2/GTBP by the
diaminocyclohexane ring present in the oxaliplatin adduct. Resolution
of this question will require detailed structural information about the
differences in cisplatin and oxaliplatin adducts complexed with
hMSH2. The current information provides a reasonable basis for
predicting that loss of DNA mismatch repair will cause resistance to
Discussion
other platinum-containing analogues that do not have substitutions on
the amine groups.
The present study indicates that loss of DNA mismatch repair due
The sequence of events in DNA mismatch repair is believed to
to lack of either hMSH2 or hMLHI activity results in resistance to
both cisplatin and carboplatin, drugs that form the same adducts in begin with the binding of hMSH2/GTBP to the mismatch, followed
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DNA MISMATCH REPAIR AND PLATINUM DRUG RESISTANCE
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+
+
antl-hMLH1
+
+
an11@hMSH2
+
+
Fig. 3. Recognition of cisplatin (A) and oxaliplatin
(B) adducts by nuclear protein extracts documented
by mobility shift assays. The 123-bp DNA fragment
+
was platinated to the same drug:nucleotide molar
.
ratio of 0.05 with cisplatin or oxaliplatin. The carrier
Cisplatintreated DNA
Nuclear Extract
anfl-Bcl-x1
+
DNA was poly(dklc)-poly(dI-dc), and the competitor
DNA was salmon sperm DNA platinated to a drug:
nucleotidemolarratioof0.05witheithercisplatin(A)
123456789
B
or oxaliplatin (B) and added in 1000-fold excess
relative to the DNA.
@
•@@ipd
‘@‘
•@11@ @.
+
+
+
4
+
+
+
+
4
+
Oxaliplatin
+
+
+
+
+
+
+
Nuclear
+
+
+
+
+
+
+
Carrier
+
Competitor
+
+
antl-hMLH1
+
1@
anli-hMSH2
+
+
+
4.
treated
DNA
Extract
antl-Actin
by the recruitment of a hMLH1IPMS2 heterodimer (23). The fact
complex fully functional for nucleotide mismatch repair is required
that deficiency in DNA mismatch repair due to loss of hMLH1
for the generation of this signal. hMLH1 has already been identi
function results in resistance to cisplatin and carboplatin suggests
fled as having a function in meiotic recombination that does not
that the hMSH2/GTBP complex by itself is not able to generate the seem to involve hMSH2 (24), and it is possible that the signaling
signal that contributes to the triggering of apoptosis and whose loss
function of the mismatch repair complex can be mediated by only
results in resistance. However, it is not clear that a complete
a subset of all of the proteins required for full DNA repair activity.
4885
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1996 American Association for Cancer Research.
DNA MISMATCHREPAIRAND PLATINUMDRUG RESISTANCE
The spectrum of mutations that result in loss of DNA mismatch
repair activity may differ from the spectrum of mutations that
produce drug resistance.
A variety of other proteins also bind to cisplatin adducts, the
best-studied of which are members of the high mobility group family
of proteins (25, 26). In the case of one of these, the Saccharomyces
cerevisiae gene Ixrl, mutational loss is also associated with low-level
resistance (27). Proteins containing the high mobility group domain
can antagonize the excision of cisplatin adducts by the nucleotide
excision repair mechanism (28). How any of these interact with the
DNA mismatch repair proteins is unkown, but it is likely that they also
play a role in the recognition of cisplatin damage and generation of an
injury signal.
How likely is it that the relatively small degree of resistance to
cisplatin and carboplatin associated with loss of mismatch repair is
clinically significant? Unlike many other drugs, only quite small
degrees of cisplatin resistance (<2-fold) are required to account for
clinical failure of treatment (2). The 2.1-fold resistance observed in
the HCT116 system and 1.8-fold resistance observed in the HEC59
system would be expected to be sufficient to result in the gradual
enrichment of cells in the tumor population deficient in mismatch
repair during repeated courses of treatment, and this may contribute to
the phenomenon of acquired cisplatin resistance that is so common
among patients with tumors such as ovarian carcinoma and squamous
cell carcinomas of the head and neck that are initially quite responsive
to this drug. The results of this study showing that loss of mismatch
repair does not confer resistance to oxaliplatin or JM216 suggest that
the use of these drugs may avoid this problem and recommend these
drugs for use in tumors deficient in mismatch repair.
8. Duckett, D. R., Drummond, J. T., Murchie, A. I. H., Reardon, J. T., Sancar, A., Lilley,
D. M. J.. and Modrich, P. Human MutSa recognizes damaged DNA base pairs
containing 0@-methylguanine, O@°-methylthymine,or the cisplatin-d(GpG) adduct.
Proc. NatL Acad. Sci. USA, 93: 6443-6447, 1996.
9. Levi,F.,Perpoint,B.,Garufi,C..Focan,C.,Chollet,P.,Depres-Brummer,
P.,Zidani,
R., Brienza, S., ItZhaki, M., Iacobelli. S., Kunstlinger, F., Gastiaburu, J., and Misset,
J-L. Oxaliplatin activity against metastatic colorectal cancer: a Phase II study of 5-day
continuous venous infusion at circadian rhythm-modulated rate. Eur. J. Cancer, 29:
1280—1284,
1993.
10. Raynaud, F. I., Mistry, P., Donaghue, A., Poon, 0. K., Kelland, L. R., Barnard, C. F.,
Murrer, B. A., and Harrap, K. R. Biotransformation of the platinum drug JM216
following oral administration to cancer patients. Cancer Chemother. Pharmacol., 38:
155—162,
1996.
11. DiSaia, P. J., Sinkovics, J. 0., Rutledge, F. N., and Smith, J. P. Cell-mediated
immunity to human malignant cells. Am. J. Obstet. Gynecol.. 114: 979—989, 1972.
12. Koi, M., Umar, A.. Chauhan, D. P., Cherian, S. P., Carethers, J. M., Kunkel, T. A.,
and Boland, C. R. Human chromosome 3 corrects mismatch repair deficiency and
microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine
tolerance
in colon tumor cells with homozygous hMLI-I1 mutation. Cancer Res., 54: 4308—
4312, 1994.
13. Boyer,J. C., Umar,A., Risinger,J. I., Lipford, J. R., Kane,M.. Yin, S.,Barrett,J.C.,
14.
15.
16.
17.
18.
hMSH2—pl6O
heterodimer that restores DNA mismatch repair to tumor cells. Science
(Washington DC), 268: 1909—1912, 1995.
19. Szymkowski, D. E., Yarema, K., Essigmann, J. M.. and Lippard, S. J. An intrastrand
d(GpG) platinum cross-link in duplex Ml3 DNA is refractory to repair by human cell
extracts. Proc. NatI. Acad. Sci. USA, 89: 10772—10776,
1992.
Acknowledgments
We thank Dr. Ming Lu for his assistance in the mobility shift assays and
Drs. C. Richard Boland, Minoru Koi, and Thomas A. Kunkel for kindly
providing the cell lines.
References
I. Kavanagh,J.. Tresukosol,D., Edwards,C., Freedman,R., Gonzalezde Leon,C.,
Fishman, A., Mante, R., Hoed, M., and Kudelka, A. Carboplatin reinduction after
taxane in patients with platinum-refractory epithelial ovarian cancer. J. Clin. Oncol.,
13: 1584—1588,1995.
2. Andrews, P. A., Jones, J. A., Varki, N. M., and Howell, S. B. Rapid emergence of
acquired cis-dichlorodiammineplatinum(ll) resistance in an in vivo model of human
ovarian carcinoma. Cancer Commun., 2: 93-100, 1990.
3. Gately, D. P., and Howell, S. B. Cellular accumulation of the anticancer agent
cisplatin: a review. Br. 3. Cancer, 67: 1171—1
176, 1993.
4. Aebi, S., Kurdi-Haidar, B., Gordon, R., Cenni, B., Zheng, H., Fink, D., Christen,
R. D., Boland, C. R., Koi, M., Fishel, R., and Howell, S. B. Loss of DNA mismatch
repair in acquired resistance to cisplatin. Cancer Res., 56: 3087-3090, 1996.
5. Fishel, R., and Kolodner, R. D. Identification of mismatch repair genes and their role
in the development of cancer. Curr. Opin. Genet. Dev., 5: 382—395,1995.
6. Kat, A., Thilly, W. G., Fang, W. H., Longley, M. J., Li, G. M., and Modrich, P. An
alkylation-tolerant mutator human cell line is deficient in strand-specific mismatch
repair. Proc. Natl. Acad. Sci. USA, 90: 6426-6428, 1993.
7. Griffin, S.. Branch, P., Xu, Y. Z., and Karran, P. DNA mismatch binding and incision
at modified guanine bases by extracts of mammalian cells: implications for tolerance
to DNA
methylation
Kolodner, R. D., and Kunkel, T. A. Microsatellite instability, mismatch repair
deficiency, and genetic defects in human cancer cell lines. Cancer Res., 55: 6063—
6070, 1995.
Rathmell, W. K., and Chu, 0. A DNA end-binding factor involved in double-strand
break repair and V(D)J recombination. Mol. Cell. Biol., 14: 4741—4748. 1994.
Andrews, P. A., and Jones, J. A. Characterization of binding proteins from ovarian
carcinoma and kidney tubule cells that are specific for cisplatin-modified DNA.
Cancer Commun., 3: 93—102,1991.
Fishel, R., Ewel, A., and Lescoe, M. K. Purified human MSH2 protein binds to DNA
containing mismatched nucleotides. Cancer Res.. 54: 5539—5542. 1994.
Palombo, F., Gallinari, P., laccarino, I., Lettieri, T., Hughes, M., D'Athgo, A.,
Truong, 0., Hsuan, J. J., and Jiricny, J. GTBP, a 160-kilodalton protein essential for
mismatch-binding activity in human cells. Science (Washington DC), 268: 1912—
1914, 1995.
Drummond, J. T., Li, G-M., Longley, M. J., and Modrich, P. Isolation of an
damage. Biochemistry,
20. Huang, J-C., Zamble, D. B., Reardon, J. T., Lippard, S. J., and Sancar, A. HMG
domain proteins specifically inhibit the repair of the major DNA adduct of the
anticancer drug cisplatin by human excision nuclease. Proc. Natl. Acad. Sci. USA, 91:
10394—10398,
1994.
21. Fraval, H. N., Rawlings, C. J., and Roberts, J. J. Increased sensitivity of UV-repair
deficient human cells to DNA-bound platinum products which unlike thymine dimers
are not recognized by an endonuclease extracted from Micrococcus luteus. Mutat.
Rca.,51: 121—132,
1978.
22. Dijt, F. I., Fichtinger-Schepman, A. M. J., Berends, F., and Reedijk, J. Formation and
repair of cisplatin-induced adducts to DNA in cultured normal and repair-deficient
human fibroblasts. Cancer Res., 48: 6058—6062, 1988.
23. Li, G-M., andModrich, P. Restorationof mismatchrepairto nuclearextractsof H6
colorectal tumor cells by a heterodimer of human Mutt. homologs. Proc. NatI. Acad.
Sci. USA, 92: 1950—1954, 1995.
24. Baker, S. M., Plug, A. W., Prolla, T. A., Bronner, C. E., Harris, A. C., Yao, X.,
Christie, D-M., Monell, C., Arnheim, N., Bradley, A.. Ashley, T., and Liskay, M.
Involvement of mouse MLII! in DNA mismatch repair and meiotic crossing over.
Nat. Genet., 13: 336—342,1996.
25. Turchi, J. J., Li, M., and Henkels, K. M. Cisplatin-DNA binding specificity of calf
high-mobility group 1 protein. Biochemistry, 35: 2992—3000, 1996.
26. Baxevanis, A. D., and Landsman, D. The HMG-l box protein family: classification
and functional relationships. Nucleic Acids Res., 23: 1604—1613,1995.
27. Brown, S. J., Kellett, P. J., and Lippard, S. J. Ixrl, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science (Washington DC), 261:
603—605,1993.
28. McA'Nulty. M. M., and Lippard, S. J. The HMG-domain protein Ixrl blocks excision
repair of cisplatin-DNA adducts in yeast. Mutat. Res., 362: 75—86,1996.
33: 4787—4793, 1994.
4886
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The Role of DNA Mismatch Repair in Platinum Drug Resistance
Daniel Fink, Sibylle Nebel, Stefan Aebi, et al.
Cancer Res 1996;56:4881-4886.
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