Download Expression of the Human Mismatch Repair Gene

(CANCER RESEARCH 56. 235-240. January 15. 1996]
Advances in Brief
Expression of the Human Mismatch Repair Gene hMSH2 in Normal and
Neoplastic Tissues'
Fredrick S. Leach, Kornelia Polyak, Marilee Burrell, Karen A. Johnson, David Hill, Malcolm G. Dunlop,
Andrew H. Wyllie, Paivi Peltomaki, Albert de la Chapelle, Stanley R. Hamilton, Kenneth W. Kinzler, and
Bert Vogelstein2
The Johns Hopkins Oncology center. Baltimore, Maryland 21231 (F. S. L, K. P., 5. R. H., K. W. K., B. V.]; Oncogene Science, cambridge, Massachusetts 02142
(M. B., K. A. J., D. H.]; Department of Surgery and MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom EH4 2XU (M. D.]; Department of
Pathology, Universiry Medical School, Edinburgh, United Kingdom EH8 9AG (A. W.]; Department of Medical Genetics, University of Helsinki, Helsinki, Finland @Xi290
(P. P., A. ci. I. ci; Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2/205 [5. R. H.]; and Howard Hughes Medical Institute
1K. P., B. V.]
Abstract
and hPMS2) have been associated with HNPCC when inherited in
mutant form (14—16).
The MMR and NER genes appear to be typical “housekeeping―
genes. They are found in every prokaryotic and eukaryotic organism
examined thus far, and their structure and function appearto be highly
Hereditarynonpolyposis
colorectalcanceris causedby inheritedmu
tations of mismatch repair genes.We developedmonoclonalantibodiesto
the prototypehuman mismatchrepair genehMSH2 and usedthem to
detect an immunoreactive protein of Mr 100,000111mismatch-proficient
cell lines.In addition,a Mr 150,000proteincoimmunoprecipitated
with conserved(2). Tumors in patients with NER defects are largely
the hMSH2 geneproductin cell linesexpressinghMSH2. Immunohisto confined to the skin. This is consistentwith the idea thatUV radiation
chemistrydemonstratedthat thehMSH2 proteinwasexclusivelynuclear. is responsible for most DNA defects for which NER is indispensable
Whereas the hMSH2 protein was expressed in a variety of tissues, the
moststrikingpatternwasobservedin esophageal
andintestinalepithelia,
whereexpressionwaslimitedto the replicatingcompartment.Neoplastic
cells within benignand malignantmismatchrepair-proficienttumors
expressedthe protein,but no hMSH2 immunoreactivitywasobservedin
the colorectaltumorsof patientswith germline!iMSH2 mutation.These
resultshaveimplicationsfor tumorigenicmechanisms
and,potentially,for
diagnosis.
Introduction
Three classes of genes causing cancer predisposition have been
discovered:(a) oncogenesthat positively regulate cell growth; (b)
tumor suppressor genes that negatively regulate cell growth; and (c)
DNA repair genes that indirectly control proliferation by limiting the
rate of mutations of growth controlling genes. The first repair genes
implicated in tumor predispositionwere thoseresponsiblefor NER3
and associated with xeroderma pigmentosum and related autosomal
recessive inherited syndromes (1). More recently, inherited mutations
of genes involved in MMR have been shown to be responsible for
HNPCC (2—4).
HNPCC is inherited in autosomaldominantfashion
and can be caused by mutations in any one of at least four genes. The
prototype human MMR gene hMSH2 (5—7)is a homologue of the
bacterial mutS gene (8). Its product binds to a second mutS homo
logue, GTBP; both together recognize mismatched basepairs in DNA
(9—1
1). The mutL gene products interact with mutS proteins to recruit
nucleases, polymerases, and other proteins required for the repair
process(8, 12, 13). Three human mutL homologues (hMLHJ, hPMSJ,
Received 9/22/95; accepted I 1/29/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by
NIH
Grants
CA35494
and
CA62924,
The
Clayton
Fund,
The Scottish Health Department, The Academy of Finland, The Finnish Cancer Societies,
The Sigrid Jusilieus Foundation, and The Folkhason Institute of Genetics. B. v. is an
American Cancer Society Research Professor and Investigator of the Howard Hughes
Medical Institute.
2 To
whom
requests
for reprints
should
be addressed,
at the Johns
Hopkins
Oncology
Center, 424 North Bond Street, Baltimore, Maryland 21231.
3 The
abbreviations
used are: NER,
nucleotide
excision
repair;
MMR,
mismatch
repair;
HNPCC, hereditarynonpolyposiscolorectalcancer;CR, colorectal;FBS, fetal bovine
serum; GTBP, GT-binding protein; IP, immunoprecipitation; mAb, monoclonal antibody.
(1). However, the organ specificity of tumors arising in HNPCC
patientsis moredifficult to explain.On the basisof their housekeep
ing nature and their apparently ubiquitous expression patterns (at least
as judged by assessmentof mRNA), one might have expected that
MMR deficiencywould be associatedwith tumorsof virtually every
renewingcell populationin the body. Nevertheless,the greatmajority
of tumors in HNPCC patients occur in the large intestine and endo
metrium, with only occasional tumors elsewhere (3, 4). Continually
renewing cell populations in the skin, blood, lymphoid organs, breast,
lung, stomach, esophagus, small intestine, and other organs rarely
develop neoplasia in these patients.
There is as yet no informationon the expression of MMR proteins
in higher eukaryotes. In the studies reported herein, we developed
mAbs to the prototype human MMR gene hMSH2 and addressedbasic
questions about its expression in normal and neoplastic tissues.
Materials and Methods
CR cancerlineswereobtainedfromAmericanTypeCultureCollectionand
grown to log phasein McCoy's 5A mediumcontaining10%FBS. Lympho
blastoidcells weregrown in RPM! 1640containing10%FBS.Sf9 cells were
grown at 27°C
in Grace'smediumcontaining10%FBS.
RecombinanthMSH2 Clones. The completehMSH2coding region was
assembledfrom overlappingcDNA clones(6). An NcoI sitewasengineeredat
theinitiating methionineusingsyntheticoligonucleotidesto producecloneE5.
An NcoI-HindHI fragmentof E5 containing the first 550 amino acids of
hMSH2 was then cloned into the NcoI and HindIll sites of the vector pGSTag
(17) to generatepGST-MSH-N.A PstI-EcoRIfragmentof hMSH2cDNA
containingthe carboxyl-terminal330 aminoacidswasclonedinto the EcoRi
and PstI sites of pGSTag to generate pGST-MSH-C.
Fusion proteins were
purified througha glutathioneagaroseresin.A baculovirusvectorcontaining
thecompletehMSH2codingregionwasproduced
by cloningthe3.1-kbNcoI
fragment
of clone E5 into the vector pBluBacHis
(Invitrogen).
Baculovirus
wasproducedfrom this vectorasdescribedby the manufacturer,andhMSH2
protein was purified with a nickel-chelating resin (Invitrogen).
mAbs. FemaleBALB/c X C57B1/6 F1 mice were immunizedby i.p.
injectionof purified pGST-MSH-Nor pGST-MSH-Cfusion proteinsin Ribi
adjuvant(Ribi ImmunochemResearch,Inc.). The fusion proteinswere cx
pressed as insoluble proteins in Escherichia coli, collected by centrifugation
after cell lysis by passagethrough a French pressure cell (SLM Instruments),
and purified by electroelution using SDS-PAGE. Hybndomas were produced
235
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EXPRESSION
@
OF hMSH2 IN NORMAL
Blot
Analyses.
IP was performed
with
extracts
0
z
w
LI@
148-
at4°C,
followed by microcentrifugation.Theappropriateantibodieswerethen
of 10
0
0
200...
from
[35S]methionine-labeled cells (200 MCi/mi, 6 h). Extracts were made as de
scribed (19) and precleared by incubation with protein A-Separose for 60 rain
added to the extracts, to a final concentration
TISSUES
.0
as described(18), exceptthat test bleedsand hybridomaswere screenedby
ELISA for anti-MSH2 reactivityusingGST-MSH-N- and GST-MSH-C
coatedmicrotiterplates.Hybridomasupematants
thatwerepositiveby ELISA
weretestedby immunoblotusinglysatesof HCT116,LoVo, andSW480cells.
Six hybridomasreactivewith the amino terminusand two reactivewith the
carboxyl terminsof hMSH2 were isolatedand subclonedtwice by limiting
dilution.
@a
and Western
AND NEOPLASTIC
@Wmi,and incubated on ice
for 4—8
h. The immunoprecipitates
werecollectedby microcentrifugationand
washed3 times, as described(19). Immunoprecipitateswere analyzedby
SDS-PAGE,and fluorographywas performedafter Enhance(Amersham)
97—
impregnation of the gels.
For Westernblots,extractsfrom unlabeledcellswerepreparedasdescribed
above.After centrifugationat 14,000X g for 5 mm in a microfuge,50 @g
of
proteinwereusedin immunoblotanalysisafter separationthrough8% SDS
PAGE. Immunodetection
usingenhancedchemilumenescence
(Amersham)
69@Ø$
was performed following the manufacturer's recommendations.
Immunohistochemistry.
Frozen tissues were obtained from surgical re
46@@
sectionsand storedat —
80°Cuntil used.Cryostatsectionswere placedon
SuperFrost
slides (Fisher) and fixed in Histochoice
(Ameresco)
for 10 mm at
room temperature, then stored in PBS at 4°Cuntil use (up to 2 weeks).
Immunohistochemicalanalysiswasperformedas described(20), exceptthat
the antibodies were used at 2
@g/ml,and slides were incubated for 2 h (tissue
culturecells) or overnight(cryostatsections)at room temperature.The ABC
method(Vector)wasusedfor detection,andsectionswerecounterstained
with
methyl green.
Fig. 2. IF analysis with mAb FEll.
Mr 150,000 pmtein (*)
Results
Characterization of Antibodies. As described in “Materials
and
Methods,―
mAbs were generated against the amino-terminal and
carboxy-terminal ends of hMSH2. Among several antibodies tested,
the carboxy-terminal-directed mAb FE1 1 (IgG1) exhibited the best
specificity and reactivity, although another COOH-terminal-specific
antibody (EH12) and two NH2-terminal-specific antibodies (GB 12
and CF7) yielded similar results to those shown in Figs. 1 and 2.
Western blot analysis revealed that FEll detected a Mr 100'000
protein in extractsfrom Sf9 cells infected with a recombinantbacu
lovirus expressinghMSH2 (Fig. 1). The migration of hMSH2 in
SDS-PAGEwasslightlyslowerthanpredictedfromits sequence
(Mr
60
—Co
(0
1@
,@‘
1-
@JI
SW480 cells were labeled with [35S}methionine,
and extractswere immunoprecipitatedas describedin “Materials
and Methods.―
The
antibodiesusedare indicatedabovethe lanes.Arrows, hMSH2 and the coprecipitated
.200kd
94,000). No immunoreactive protein was present in control baculovi
ms-infectedSf9 cells (data not shown).MMR-proficient mammalian
cells (e.g., SW480, Ref. 21) also contained an immunoreactive
polypeptidethatmigratedat theidenticalposition.TheLoVo cell line,
which contains a homozygous deletion of hMSH2 (22, 23), had no
immunoreactive protein (Fig. 1). The HCT116 line is MMR deficient
(21) due to a homozygous nonsensemutation of hMLHJ but contained
a normal-sized hMSH2 protein (Fig. 1). This result demonstrated that
the synthesis and stability of hMSH2 is independent of the hMLH1hPMS2 complex that interacts with it functionally (12, 13) because
HCT1 16 lacks this complex (12).
We next determined whether the mutant hMSH2 gene products
found in HNPCC patients could be detected by immunoblot. K. K. is
an HNPCC patient with a germline splice site mutation at the 3' end
of exon 5 (24), resulting in an in-frame deletion of codons 265—3
14.
The Mr 85,000 protein predicted to result from this mutation was not
evident in the immunoblot prepared from EBV-transformed lympho
blasts of K. K. Only the full-length protein, presumably produced
from the unaffected allele, was observed (Fig. 1). Similarly, only
full-length proteins were evident in four other lymphoblastoidcell
lines from patients whose hMSH2 genes were predicted to contain
abnormal, short polypeptides, when tested with FEll or amino
terminal-specific mAbs (data not shown). These data suggest that
aberrant hMSH2 polypeptides or the mRNAs encoding them were
unstable.
hMSH2@'-
_.69kd1
Fig. 1. Immunoblot analysis with mAB FEll. Extracts of insect cells infected with
MSH2-expressing baculoviruses (Baculo. ); of human CR cancer cell lines SW480, LoVo,
andHCT 116;or of the K. K. lymphoblastoidcell lines wereappliedto SDS-polyacryl
amidegels,blotted,andprobedwith mAB FE11.LoVo cellscontaina deletionof MSH2
anddo not expresshMSH2 mRNA. K. K. is anHNPCCpatientwith anin-framedeletion
of exon 5 within the hMSH2 mRNA; the Mr 85,000protein predictedfrom the cDNA
sequencecannot be detected, indicating instability of the protein (see text).
IP experimentswerethenperformedwith radiolabeledcell extracts.
Under the conditions used, several polypeptides were precipitated
with mAb FEll, but only two proteins of 100 and 150 kb were
specifically immunoprecipitated (Fig. 2). This specificity was con
firmed with control antibodies (e.g., Fig. 2) and by analysis of other
cell lines; three cell lines with wild-type hMSH2 genes (HCT116,
RKO, SW480) each synthesizedMr 100,000 and 150,000 polypep
tides immunoprecipitated with FE! 1, whereas neither of these
polypeptides was observed in extracts of the hMSH2-deleted LoVo
cell line (datanot shown).
236
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1996 American Association for Cancer Research.
@
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-@I
a
EXPRESSION
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.‘@
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.
I;@
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,
@
.-..
,
,
OF hMSH2 IN NORMAL
-,,
-. .
-
‘—@‘@
with FE1 1 re
90% of unsynchronized SW480 cells in culture were intensely reac
tive, indicating that hMSH2 expression was largely independent of the
cell cycle phase. The chromosomes were stained during mitosis,
suggesting a relatively stable interaction of hMSH2 with chromatin.
The hMSH2 protein was expressed in a variety of human tissues,
::@
‘j'.
Immunocytochemistry
vealedthat the hMSH2 proteinwasexclusivelynuclear(Fig. 3). Over
‘
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TISSUES
Immunohistochemistry.
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including thyroid,heart,smooth muscle, and the germinalcenters of
lymphoid follicles. Staining was particularly prominent in the epithe
hum of thedigestivetract,extendingfrom esophagusto rectum.In the
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decreasedas cells migratedthroughthe pricklezone anddifferentiated
.
@:
(Fig. 4, A and B). In the stomach and small and large intestines,
reactivity was confined to the epithelium of the bottom half of the
crypts (e.g., Fig. 4, C and D). In colonic neoplasms, this compart
-@,,
@:‘
‘@
@ A
.-.-@
mentalizationwaslost becausecells throughoutthe neoplasticglands,
extending to the luminal surface, stained intensely. From 50 to 90% of
the epithelial cells from each of 10 CR adenomas and 8 CR carcino
mas reacted strongly with FE1 1 (e.g., Fig. 4, E and F, and Fig. 5, A
and B). Staining of adjacent sections with the Ki67 antibody revealed
a similar but somewhat less impressive extension of the replicative
compartment (data not shown).
,-‘
P
,a@ ‘
“
‘@‘
“@‘
@•)@R@
Is
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%
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esophagus, high levels of hMSH2 expression were observed in the
baseal zone closest to the basementmembrane, and staining gradually
@S
Specificity in immunohistochemicalexperiments is often difficult
to substantiate. The immunohistochemical specificity of FE1 I was
supported by the patterns observed in CR tumors from two HNPCC
patients with germline hMSH2 mutations resulting in COOH-terminal
‘I@
•‘..,@
. S
.1
@•!•.
•
truncations(23). It hasbeenshownthat in tumorsarising from such
patients,the wild-type /iMSH2 allele inherited from the unaffected
parent is usually inactivated(23, 25). In eachof four tumors from
these patients (an adenoma and carcinoma from each), no staining
t
.@,
with FE! 1 was observed (e.g., Fig. 5, C and D). A subset of the
“p1
nonneoplasticstromalcells within the samesectionreactedwith the
antibody (Fig. 5, C and D), and neoplastic epithelial cells stained with
the Ki67 antibody (not shown), providing appropriate controls. In
contrast, two tumors from patients with truncating mutations of
hMLHJ stained normally with FE! 1, as did all 18 CR tumors from
patientswithout HNPCC notedabove.
t,
Discussion
B@,
@ r wte@.@
@
.@IlI
•
•
@
O
The results described herein demonstrate that hMSH2 is a widely
expressedprotein. Its expression in normal intestinal tract epithelium
was limited to the replicative compartment, suggesting transcriptional
or translational control analogous to other proteins involved in DNA
replication. The nuclear localization of FZMSH2demonstrated here
may be due to potential nuclear localization signals at codons 871—
882 and 907—918(6). The hMSH2 protein migrates slightly slower
than that predicted from its sequence, both in natural form in mam
malian cells and in recombinant form in insect cells (Fig. 1). Whether
this difference in mobility representsposttranslational modification or
.,@
..
;1,@
@:
unusualsecondarystructureis not known.
The hMSH2 protein is apparently complexed with another Mr
•@rr
150,000 protein
(Fig. 2). This likely
represents the GT mismatch
binding protein GTBP identified by Palombo et a!. (9) and Drummond
et a!. (10). GTBP was shown to copurify
1.'
@
,@.
C
.
with hMSH2,
suggesting
that
the two proteins may exist as a heterodimer (10). Our IP data support
this suggestion.
-.
, -‘é@è)@
4@-
Fig. 3. Immunocytodetection of the hMSH2 protein. SW480 cells growing in culture
were prepared for immunohistochemistry as described in “Materials
and Methods.―
Cells
in A were stained with preimmune IgG; cells in B and C' were stained with FEI I. Strong
nuclear staining is observed in B and C'. A and B, X 100; C, X200.
‘
237
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1996 American Association for Cancer Research.
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EXPRESSION
OF hMSH2 IN NORMAL
AND NEOPLASTIC
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Fig. 4. Immunohistochemistry of human tissues. Cryostat sections of esophagus (A and B), small intestine (C and D), and a villous adenoma of the colon (E and F) were stained
with mAB FE1 1 as described in “Materials
and Methods.―
Compartmentalized staining (black nuclei) is noted at the basal zone of the esophageal squamous cell epithelium (A and
B) and crypt epithelium of the small intestine(C and D). In contrast,stainingof virtually all epithelial cells is observedin the adenoma(E and F). The sectionswere counterstained
with methyl green. A and D, X 100; B, X400; C and E, X20. @,
basal layer of epithelium.
Approximately one-half of the hMSH2 mutations identified in
HNPCC patients result in a substantially altered molecular weight of
the predicted protein. We were hopeful that these altered proteins
could be detected using specific mAbs, thereby facilitating presymp
tomatic diagnosis. Unfortunately, the mutant gene products tested
seemto be unstable,either at the RNA or protein level. Instability of
truncated proteins has many precedents. In particular many of the
truncated APC proteins found in familial adenomatouspolyposis
patients are also undetectable by Western blot analysis (18).
Although immunoblot analysis did not prove useful for diagnosis,
238
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1996 American Association for Cancer Research.
@
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EXPRESSION OF hMSH2 IN NORMAL AND NEOPLASTIC TISSUES
A
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Fig. 5. Immunohistochemistry of CR cancers. In the CR carcinoma with normal mismatch repair, most nuclei react with mAb FE1 I, as do scattered nonneoplastic cells in the stroma
(A andB). In the cancerfrom the HNPCC patientwith an hMSH2 mutation(Cand D), only a portionofthe nonneoplasticstromalcells(5) reactwith the antibody,whereastheepithelial
cells (E) are totally unstained. A and C, X 100; B and D, x400. Nonreactive nuclei are green becauseof the methyl green counterstain used.
the inmiunohistochemical
results were promising. At least four dif
ferent genes can cause HNPCC when inheritedin mutantform, and
the mutational spectrum within each gene is wide (3). This poses a
tremendous diagnostic challenge, and screening each of these genes
for mutations would be impractical. Simple methods to indicate which
gene was inactivated in an individual kindred with HNPCC would
considerably simplify the diagnostic strategy. The wild-type copy of
MMR genes, inherited from the unaffected parent of an HNPCC
3, Marro, G., and Boland, R. J. Hereditary nonpolyposis colorectal cancer: the syn
drome, the genes, and historical perspectives. J. Nail. Cancer Inst., 87: 1114—1
125,
1995.
4. Lynch, P. M., Cavalieri, R. J., and Boland, C. R. Genetics, natural history, tumor
spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated
review., Gastroenterology, 104: 1535—1549,1993.
5. Fishel, R., Lescoe, M. K., Rao, M. R. S., Copeland, N. G., Jenkins, N. A., Gather, J.,
Kane,M., andKolodner,R. The humanmutatorgeneMSH2andits associationwith
6.
patient,is usually inactivatedin tumors(23, 25, and referencesthere
in), leaving the cell with no functional protein and, in particular, no
proteincontainingcarboxyl-terminalepitopes.Our resultsareconsist
ent with the idea that staining with antibodies specific for the carboxyl
terminus of MMR gene products could thereby provide information as
to the culprit gene. Once antibodies to hMSH2 and the other MMR
proteins that are reactive with formalin-fixed, paraffin-embedded
samples become available, it will be possible to test this hypothesis in
a larger number and variety of patients and to determine the utility of
immunohistochemistry as an adjunct to genetic diagnosis.
7.
8.
9.
hereditary nonpolyposis colon cancer. Cell, 75: 1027—1038,1993.
Leach, F. S., Nicolaides, N. C., Papadopoulos. N., Liu, B., Jen, J., Parsons, R.,
Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X-Y., Zhang,
J., Meltzer, P. 5., Yu, J-W., Kao, F-T., Chen, D. J., Cerosaletti, K. M., Fournier,
R. E. K., Todd, S., Lewis, T., Leach, R. J., Naylor, S. L., Weissenbach,J., Mecklin,
J-P., Jarvinen,H., Petersen,G. M., Hamilton, S. R., Green, J., Jass,J., Watson, P.,
Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B.
Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 75:
1215-1225,1993.
Palombo,F., Hughes,M., Jiricny, J., Truong, 0., and Hsuan,J. Mismatch repair and
cancer. Nature (Lond.), 367: 417—418,1994.
Modrich, P. Mechanisms and biological effects of mismatch repair. Annu. Rev.
Genet., 25: 229—253,1991.
Palombo,F., Gallinari, P., Iaccarino,I., Lettieri,T., Hughes,M.. D'Arrigo, A., Truong,
0., Hsuan,3. J., and Jiricny,J. GTBP, a 160-kilodaltonproteinessentialfor mismatch
binding activity in human cells. Science (Washington DC), 268: 1912, 1995.
10. Drummond,J. T., Li, G., Langley, M. J., andModrich, P. Isolationof an hMSH2-
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Expression of the Human Mismatch Repair Gene hMSH2 in
Normal and Neoplastic Tissues
Fredrick S. Leach, Kornelia Polyak, Marilee Burrell, et al.
Cancer Res 1996;56:235-240.
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