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(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 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1996 American Association for Cancer Research. 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. @ @ @ •1@ -@I a EXPRESSION @ . •@ @ .@: .@ “ .‘@ .@ . I;@ @ , @ .-.. , , 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 ‘ t@;-; ,: TISSUES Immunohistochemistry. @@:‘Ti:-;@.;J', @. AND NEOPLASTIC @.:.; @ ., --:@ ‘.‘ ‘@ r'@ ?_#‘. @ . 1@ @ @ .. I' . ¶@ @--.-@:.--@ ., ,<;. “ @.. .. -.@ 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 @ ‘.. @ .@ . ,@ @ . .‘ .. @.. ,. @ .‘, @. ... . . (/tô@@@@ @ , . . ,r ,- ‘ @ . / - 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 .t,@ *. % @ :‘@@‘ ..@,- 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. @ .@ EXPRESSION OF hMSH2 IN NORMAL AND NEOPLASTIC @: TISSUES B A @ p @ •i$@@ @• @ i@. * . @$11@1@q,@ 0@ * . •r' . . . @, @ .—@ . @a -‘@ 4 * 0 @‘ ;@,. ,. . @4 4, @ m :@# ::;‘ __*. ‘@0; f@S―.' D 1@ @- .@ ..,,@ @ . @ ,..--... , (:@. :.@:‘4;@ @ . : “)fr F E @ ,@-\ @ . :‘@ @“ .:@ @ @ “@@—‘:‘:@:@ ;<2@ .. ‘ “I ..I@ ::@ @ @ $ --:-“ .@-‘ @ - , . ‘-:â€-̃‘ @ ‘,— @ (‘ . ‘ @ \ ; ‘@ @4 -. .. ‘@ --- ‘,—- . :.@ @-. - : ..@J- @ @ :@T: ., .@_;--.-,. .j., p 3d L@:@ -,@ A 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. @ t@ S S@* EXPRESSION OF hMSH2 IN NORMAL AND NEOPLASTIC TISSUES A @ @‘; .) @ .T@T'@;: C D ,. E\ , Js If @ , . . •@@,@:‘..‘. • - • , ‘ø ‘j 5 E@ @. @ I *@e• , - ,..@ E54 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. 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Leach, Kornelia Polyak, Marilee Burrell, et al. Cancer Res 1996;56:235-240. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/2/235 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1996 American Association for Cancer Research.