[CANCER RESEARCH57, 1673-1677. May I. 19971

Advances in Brief

Reduced Expression of Mismatch Repair Genes Measured by Multiplex ReverseTranscription-Polymerase Chain Reaction in Human Gliomast

Qingyi Wei,2 Melissa L. Bondy, Li Mao, Yongli Guan, Lie Cheng, Joan Cunningham, Youhong Fan,Janet M. Bruner, W. K. Alfred Yung, Victor A. Levin, and Athanassios P. Kyritsis

Departments of Epidemiology [Q. W., M. L B.. 1'. G., L. C., J. C.], Thoracic and Head and Neck Medical Oncology IL M., Y. F.], Pathology If. M. B.], and Neuro-OncologyIW. K. A. Y., V. A. L. A. P. K.]. The University of Texas M. D. Anderson Cancer Center, Houston. Texas 77030

Abstract

Microsatellite instability (MIN) is frequently observed in hereditarynonpolyposis colon cancer and In other sporadic cancers including gliomm. Abnormalities in at least one of five mismatch repair (MMR) genesare Implicated in the development of cancers in hereditary nonpolyposiscolon cancer and the associated MIN. Using a newly developed multiplexreverse transcription-PCR assay, we evaluated the expression of the fiveknown human MMR genes (hMSH2, hMLHJ, hPMSI, hPMS2, and GTBP)

in human gliomas by measuring simultaneously the relative levels of thetransciipts. The fi-actin gene was used as an internal control for RNAdegradation and DNA contamination and as a refrrence for quantifyingthe levels of their transcripts. Of the 33 gliomas examined, 42% (14) hadlow expression of hMSH2 (at least 4—5-foldlower than normal mean),21% (7) had low expression of hMLHJ, and 18% (6) had low expressionof hPMSJ comparedwith the expressionin the lymphocytesfrom 13normal individuals. Furthermore, six of the 33 (18%) tumor samples haddecreased expression of more than one MMR gene. Two of these sixpatients with multiple gene abnormalities had second primary cancers,and an additional patient had multifocal gliomas. Further molecularanalysis of available DNA samples indicated that one of five of thosetumors with aberrant expression of MMR genes had MIN, as comparedwith none of five tumors with normal expression. These data suggest thatreduced expression ofMMR genes is frequent in human gliomas and thataberrant expression of more than one MMR gene may be associated withincreased risk of second primary malignandes in glioma patients.

Introduction

MIN3 is a hallmark of HNPCC (1) and a common genetic alterationobserved in many malignancies, including gliomas (2) and lung (3),colon (4), bladder (5), pancreas (6), stomach (6), and endometriumcancers (7). Defective MMR is believed to be responsible for MIN.Five genes are known to participate in MMR in humans (8—16).Although the molecular mechanisms of MIN in sporadic cancers arenot well understood, germ-line mutations have been identified in atleast four MMR genes (hMLHJ, hMSH2, hPMSJ, and hPMS2) in 70%of HNPCC patients (17). Decreased MMR may be responsible for theMN observedin varioussporadiccancersas well. Mutationsin MMRgenes, however, are relatively rare in sporadic cancers; for instance,only 1 in 10 patients with sporadic colon cancer exhibiting MIN has

Received 12/31/96; accepted 3/20/97.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by a grant from the Charlotte Geyer Foundation (to

M. L. B.), by NIH Grants CA70242 and CA70334(to Q. W.), CA 70917 (to M. L. B.) andCA 55261 (to V. A. L), and by National Cancer Institute Core Grant CA16672 to M. D.

Andeson Cancer Center.2 To whom requests for reprints should be addressed, at Department of Epidemiology,

Box 189,The Universityof Texas M.D. AndersonCancerCenter, 1515HolcombeBlvd., Houston, TX 77030. Phone: (713) 792-3020; Fax: (713) 792-0807; E-mail:qwei@request.mda.uth.tmc.edu.

3 The abbreviations used are: MN, microsatellite instability; HNPCC, hereditary

nonpolyposis colon cancer; MMR, mismatch repair; RT-PCR, reverse transcription-PCR;xP, xerodermapigmentosum;dNTP,deoxynucleotidetriphosphate.

detectable mutations (18). Lack of MMR gene expression can occur inHNPCC without detectable mutations (17), suggesting that there areother mechanisms of gene inactivation. In addition, there are nomutation hot spots in the MMR genes, making detection of mutationsdifficult. In human gliomas, decreased MMR may also be involved in

tumor progression, as evidenced by increased frequency of MIN inhigh-grade tumors (2). In this study, we used a multiplex RT-PCR

assay to test the hypothesis that the expression of MMR genes isfrequently reduced in human gliomas. We found that decreased MMRgene expression was common in gliomas and was correlated withtumor progression and increased risk of second primary tumors.

Materials and Methods

Cell Lines and Tissues. We used three types of cell lines in this study:lymphoblastoid cell lines, which included three normal lines (GM00892B,GMOO131A, and GM03798B); and three nucleotide excision repair-deficientxP lines [GM02345B(group A); 0M02246B (group C), and GM02485B(group D)J, obtained from the Human Genetic Mutant Cell Repository (Camden, NJ); transformed lymphocytes (3402p, 35LIp, and 3585p) from threenormal donors (19); and four colon cancer lines that have deficient MMR andmutations in MMR genes LRef.20; LoVo (229-CCL), which has mutations inhMSH2; DLD-l (22l-CCL) and HCT-15 (225-CCL), which have mutations inGTBP; and SW48 (23l-CCL), which has mutations in hMLHIJ. These coloncancer cell lines were obtained from the American Type Culture Collection(Rockville, MD). The lymphoblastoid cells were cultured in RPM! 1640supplemented with 15% fetal bovine serum. All the other cell lines werecultured under the conditions suggested by the providers at 37°Cin a 5% CO2atmosphere. Frozen peripheral lymphocytes from 13normal blood donors werealso used. The blood samples were obtained in heparinized Vacutainers (Becton Dickinson, Franklin Lakes, NJ) and processed within 8 h for isolation andcryopreservation of lymphocytes as described previously (21).

Thirty-three glioma samples were collected at surgery. One of us (J. M. B.)examined sections of the frozen tumor blocks to document the presence ofsolid tumor free of contamination by normal or necrotic tissue. Sequentialfrozen sections were processed for mRNA isolation and cDNA synthesis. Ofthe 33 gliomas, 12 were anaplastic astrocytomas and 21 were glioblastomas.

Multiplex RT-PCR. To amplify the five MMR genes, we used a modification of our previously described multiplex RT-PCR technique (22) that willbe described in detail elsewhere.4 Briefly, by using Tri-Reagent, an RNA/DNA/protein isolation reagent (Molecular Research Center, Inc., Cincinnati,OH), and the manufacturer's protocol (23), we isolated total RNA. Then,cDNA was synthesized by reverse transcription of 0.5 @xgof random primers(Promega Biotech, Inc., Piscataway, NJ), 200 units of Moloney murine leukemia virus reverse transcriptase (United States Biochemical Co., Cleveland,

OH), 1 ,.tg of total cellular RNA, 4 pJ of 5X reverse transcriptase buffer [250mM Tris-HCI (pH 8.3), 375 misi KCI, 50 mi@i Dli', and 15 mai MgCI,; Life

Technologies, Inc., Gaithersburg, MD], 0.05 mM each dNTP, 20 units ofRNasin (Promega Biotech), and 6.5 @xlof diethylpyrocarbonate-treated water.

The 20-pi reaction mixtures were incubated at room temperature for 10 mmand 42°Cfor 45 mm, heated to 90°Cfor 10 mm, and then quickly chilled onice.

4 Y. Guan, L. Cheng. and Q. Wei. submitted for publication.

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MISMATCHREPAIRGENES IN GLIOMAS

To select the primers, we used GenBank Sequence Data Library mRNA orcDNA sequences for hMSH2 (GenBank accession no. U039l 1), hMLHJ(U07418), GTBP (U28946), hPMSI (U13695), hPMS2 (Ul3696), and f3-actin

(X0035l), accessed through the Genetic Computer Group Genetic Database(GCG version 8.0). The primer sequences used were: @3-actin,5'-ACACTGTGCCCATCTACGAGG-3' (sense) and 5'-AGCIGGCCGGACTCGT

CATACT-3' (antisense); hMSH2, 5'-GTCGGCTItGTGCGCTfC1Tf-3'(sense) and 5'-TCTCTGGCCATCAACTGCGGA-3' (antisense); !ZPMS2, 5'-

TGCATGCAGCGGA1TfGGAAA-3' (sense) and 5'-GAACCCCTCA

GAATCCACGGA-3' (antisense); GTBP, 5'-CCCTCAGCCACCAAAGAAGCA-3' (sense) and 5'-CTGCCACCACUCCTCATCCC-3' (antisense);hMLHJ. 5'-GTGCTGGCAATCAAGGGACCC-3' (sense) and 5'-CACGGT

TGAGGCATFGGGTAG-3' (antisense); and hPMSJ, 5'-GCGGCAACAGTTCGACTCC1T-3' (sense) and 5'-AGCCUGATACCCTCCCCGTF-3' (an

tisense). To select these primers, we used the strategy described previously(22), and all of the primers were synthesized by Life Technologies, Inc.

Each 50 @xlof PCR contained 3 @tlof reverse transcriptase reaction mixture,1X PCR buffer [500 mMKCI, 100 mMTris-HCI (pH 9.0), 1% Triton X-l00,and 2.5 mMMgC12J,0.04 mMeach dNTP, 2 units ofTaq polymerase (PromegaBiotech) and 25 @Mf3-actinprimers, 125 @MhMSH2 primers, 50 @MhPMS2primers, 30 @MGTBP primers, 20 @MhMLHJ primers, and 25 @MhPMSJ

primers. The mixtures were amplified in the TwinBlock Ericomp DNA amplification system (SC!, San Diego, CA) by an initial denaturation step at 95°C

for 5 mm; 29 cycles of denaturation at 95°Cfor 30 s, primer annealing at 59°Cfor 30 s, and extension at 72°Cfor 45 s; and a final elongation step at 72°Cfor10 mm. This optimal protocol allowed the amplification of all genes simultaneously in 29 cycles and gave very consistent results in repeated assays.

The RT-PCR products were separated by 1.5% agarose gel electrophoresis,stained with 0.5 @xg/mlethidium bromide, and visualized with UV light. Thebands on the photos were scanned as digitized images, and the areas of thepeaks were calculated in arbitrary units by densitometric analysis with aDigital Imaging System (Model IS-l000; a Innotech Co., San Leandro, CA).We evaluated its expression by using the internal standard (@-actin)in eachreaction as the baseline gene expression of that sample to calculate the relativevalue for each of the target genes amplified in that reaction. These values werethen compared across the samples tested. The absence of bands was verified byrepeating the multiplex PCR assays.

Microsatellite Analysis. We used six microsatellite markers: D3S1285,D9S171, D11S899, D13S133, D14S51, and DJSSIJ7 (ResearchGenetics,

Huntsville, AL). One of the primers for each marker was end-labeled with[-y-32P]ATP(4500 Cilmmol; ICN Biomedicals, Costa Mesa, CA) and T4 DNApolynucleotide kinase (New England Biolads, Beverly, MA). The PCR reac

tions were performed in a l2.5-jxl volume containing 20 ng of genomic DNA,1%DMSO, 200 LMdNTP, 1.5 mt@iMgCI2,0.4 ,.@MPCR primers (including 0.1p.M y-32P-labeled primer), and 0.5 units of Taq DNA polymerase (Life Tech

nologies, Inc.). The DNA was amplified for 35 cycles at 95°Cfor 30 s,52—60°Cfor 60 s, and 70°Cfor 60 s in a temperature cycler (Hybaid;Omnigene, Woodbridge, NJ) in 500-@xlplastic tubes, followed by a 5-mmextension at 70°C.The PCR products were separated on a 6% polyacrylamideurea-formamide gel, which was then autoradiographed. MIN was determinedby the appearance of clearly novel alleles that were not observed in pairednormal tissue controls.

Results

That our stock reaction mixture contained the optimal concentrationfor each pair of primers was confirmed by amplifying each gene

separately with the (3-actin gene only in a normal cell line and then

amplifying all five MMR genes with f3-actin in a single reaction (Fig.1A, Lanes 1—5).Because multiplex PCR of all of the five target genesand 13-actin produced the same bands as separate amplification of eachtarget gene and /3-actin (Fig. 1A, Lanes 6), we concluded that thereaction mixture was adequate for maximum amplification of eachtarget gene and that there was no apparent primer dimerization orcompetition due to primer sequence homology.

To further confirm the adequacy of our reaction mixture, we performed the RT-PCR assays on four colon cancer cell lines with known

mutations in one of the target genes to define aberrant expression in

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Fig. 1. A, control multiplex RT-PCR coamplification of five MMR genes in a normallymphoblastoid cell line (GMOO131A). M, molecular weight marker (4X174 RF DNA!HaeIll); Lane I, hMSH2 coamplified with @-actin;Lane 2, hPMS2 coamplified with@3-actin;Lane 3, GTBP coamplified with @-actin;Lane 4, hMLHI coamplified with13-actin; Lane 5. hPMSI coamplified with @-actin;Lane 6. all five MMR genes coamplifled with @-actin.B, control multiplex RT-PCR coamplification of five MMR genes and

@-actinin lymphoblastoid and colon cancer cell lines. Marker, 4X174 RF DNA!HaeIll;Normal, normal lymphoblastoid cells (GMOO13IA); XP-C. XP-C cells (GM02246B);HCT-I5. colon cancer cells; LoVo, colon cancer cells; DLD-1, colon cancer cells; SW48,colon cancer cells. The PCR products were electrophoresed in 1.5% agarose gels, stainedwith ethidium bromide, and visualized with UV light.

the test samples. As shown in Fig. 1B, normal cells (GMOO131A) andnucleotide excision repair-deficient XP-C cells (GM02246B) hadsimilar (and therefore normal) expression levels of all five MMRgenes under the assay conditions used. However, LoVo and SW48cells had no detectable expression of hMSH2 and hMLHJ, respectively, because those two cell lines have deletions in those genes (20,24). HCT- 15, DLD- 1, and SW48 appeared to express less GTBP than

normal cells did under the same assay conditions. Both HCT-!5 andDLD-l (but not SW48) cells have mutations in GTBP (16, 20). Theseresults, therefore, provided evidence that our multiplex RT-PCR assaymay help detect some genetic alterations that greatly reduce theexpression of the affected genes at the transcriptional level. Usingthese results as the criteria for aberrant expression, we performedmultiplex RT-PCR assays with the same primer cocktail on gliomasamples and normal cells.

Because we used 29 cycles of PCR to minimize the plateau effectof amplification, the definition of aberrant expression was stringent.As shown in Fig. 2, the intensity of each band in a negative of aphotograph of the agarose gel was analyzed by densitometry andquantified in arbitrary units. Only those bands the relative intensities

of which were at least four to five times less than the mean value fromnormal cells assessed under the same conditions were considered to

show reduced expression. For example, the hMSH2 peak in Lane 7(Fig. 14) was not considered aberrant, but the missing hMSH2 bandsin Lanes 2 and 3 were. This was confirmed by repeating the multiplexPCR. Therefore, expression of all five MMR genes was detectable (ornormal) by densitometric analysis in all three nucleotide excisionrepair-deficient XP cell lines (Fig. 3), six transformed cell lines fromnormal individuals (Fig. 3), and peripheral blood lymphocytes from

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B. The missing !IMSH2 bands in Lanes 2 and 3.indicated by the arrows in B, had no discerniblepeaks in A; therefore, they were considered aberrant; but the hMSH2 band of Lane 7, despite itsapparently reduced intensity, gave a discerniblepeak not more than 5-fold less than that of normalcells (Lane I in A); therefore, it was not deemedaberrant. B, photographic negative of an agaroseelectrophoresisgel showingMMRgeneexpressiondetected by multiplex RT-PCR in human gliomasamples. M, molecular weight marker (4Xl74 RFDNA!Haelll); Lane I, normal lymphoblastoid cells(GMOO131A); Lanes 2—7,glioma samples. The cx

tra small bands (less than 50 bp) in Lanes 1. 6, and7 may be primer dimers or may be due to nonspecific binding of the primers and subsequent amplification.

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13 normal individuals (Fig. 3), although the transformed normallymphocytes tended to have two to three times more mRNA for all ofthe genes tested than did peripheral blood lymphocytes (data notshown). However, reduced relative expression levels of three MMRgenes, hMSH2, hMLHJ, and hPMSJ, were observed in gliomas(Fig. 2A).

Because tumor cells are transformed, it is valid to compare themwith established cell lines. In general, glioma tumor cells had significantly lower (P < 0.05) mean expression of hMSH2 (17.6 ±15.2),hMLHJ (27.9 ±19.9), and hPMSJ (33.8 ±23.6, although a largerange of expression) than did the established cell lines (37.7 ±8.7,60.4 ±13.4, and 64.0 ±20.6, respectively) and a significantly lower(P < 0.05) mean expression of hMSH2 than normal peripheral (unstimulated) lymphocytes (3 1.1 ± 14.3) (Fig. 3), but glioma andnormal cells had similar distributions of GTBP and hPMS2 expression(data not shown). Overall, 42% (14 of 33) of the glioma samples hadgreatly reduced expression of hMSH2, 21% (seven of 33) had reduced

expression of hMLHJ, and 18% (six of 33) had reduced expression ofhPMSJ, whereas none of the samples of normal lymphocytes hadaberrant expression of any of the MMR genes. Six of the 33 (18%)patients' tumor samples had greatly reduced expression of more thanone MMR gene, and three of the six patients had had second primarycancers at other sites or were diagnosed with multifocal gliomas.Although 27% of all the patients (9 of 33) had had prior treatment,only 21% (3 of 14) of the patients who had reduced expression of atleast one MMR gene had received prior treatment. Only one of thesesix patients with reduced expression of more than one MMR gene hadreceived radiotherapy, and none of the three patients with second

cancers or multifocal gliomas had had prior treatment. Therefore, it isunlikely that the observed reduction in gene expression was influenced by chemotherapy or radiotherapy.

Although Northern blotting and sequencing analysis were not performed because of the limited amount of tumor tissue available, wewere able to examine MIN by using six microsatellite markers andpaired samples of genomic DNA (lymphocyte and tumor tissue) from10 patients, 5 with normal expression and 5 with aberrant expressionof MMR genes as detected by the multiplex RT-PCR assay. Positive

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Fig. 3. Distribution of relative expression of the MMR genes in gliomas and normalcells. AA, anaplastic astrocytomas; GBM. glioblastomas; NC, normal lymphoblastoidcells; NI., normal peripheral lymphocytes; XP, XP lymphoblastoid cells. The glioma cells(AA and GBM) had a greater range of relative expression of hMSH2, hMLHJ. and hPMSIthan normal cells (NC and NL) and XP cells.

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MISMATCHREPAIRGENES IN GLIOMAS

expression of MMR genes was relatively low. Interestingly, one oftwo patients who developed second primary tumors had both reducedexpression of more than one MMR gene and MIN, suggesting thatdecreased MMR capacity may increase the risk of developing braintumors. In a recent report on expression and mutations of MMR genesin 28 HNPCC tumors (33), 19 had MIN, 8 had mutations in eitherhMSH2 or hMLHJ, and 7 of 11 with MIN but without mutations didnot have expression of the hMSH2 and hMLHJ gene products asmeasured with a mouse monoclonal antibody.

The results of multiplex RT-PCR may not correlate with proteinexpression, because even if mutations or deletions lead to truncated,nonfunctional proteins, their mRNAs may still be detectable by RT

PCR by increasing the number of PCR cycles (8—16,20). However,even immunohistochemical methods may not always detect all mutantproteins (34). The low frequency of MIN observed in human gliomaswith aberrant MMR gene expression suggests that MMR may functionadequately with relatively low levels of gene products. Nevertheless,

the multiplex RT-PCR method has several advantages: (a) it is aquick, easy way of simultaneously screening several genes that maybe involved in a disease; (b) it can be performed with a small amountof tissue; and (c) it has an internal control, because multiple transcriptsare amplified simultaneously and, therefore, the absence of expressionof one target gene cannot be due to RNA degradation if the othergenes in the same reaction are amplified. This assay may, therefore, beuseful for rapid initial screening for the status of MMR genes ingliomas. Reduced MMR capabilities may contribute to acquired resistance to chemotherapeutic agents such as cisplatin (35, 36). In suchcases, evaluating MMR genes in tumors by multiplex RT-PCR couldhelp predict response to aggressive chemotherapy. Studies of a largecohort of patients with gliomas should be conducted to evaluate thecorrelation between this rapid MMR gene expression assay and response to treatment and survival.

Acknowledgments

We thank Drs. Bert Vogelstein and Kenneth Kinzler for providing thecDNA clones for hMSH2, hMLHJ, !ZPMSI,and hPMS2; Dr. Josef Jiricny forproviding the cDNA clone for GTBP; Dr. T. C. Hsu for providing 3402p,3Sl3p and 3S8Sp cells; Drs. Reuben Lotan and Margaret Spitz for theirthoughtful discussion and suggestions; Dr. Maureen Goode (Department ofScientific Publications) for editorial assistance; and Joanne Sider and JoyceBrown for preparation of the manuscript.

References

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NTNT

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D9S171 D14S51 D15S117Fig. 4. MIN in a glioma sample with decreased expression of hMSH2. hMLHI. and

hPMSJ as shown in Fig. 2, Lane 3. N, normal DNA; T, tumor DNA. Arrows, shiftedbands. The microsatellite markers used are shown under each panel.

MIN (Fig. 4; indicated by at least two of six markers) was identifiedin one of five patients who had aberrant expression of JZMSH2,hMLHJ, or hPMSJ but not in the other five patients who had normalexpression of the MMR genes. These results, although from a limitednumber of samples, provide evidence that the multiplex RT-PCRmethod may be useful in screening for gliomas that may have aberrations of MMR.

Discussion

In this study, by using a newly developed multiplex RT-PCR assayfor the expression of all five known human MMR genes, we demonstrated that in human gliomas, reduced expression of hMSH2 wasmost frequent, hMLHI was relatively frequent, and hPMSJ was lessfrequent, whereas GTBP and hPMS2 expression was apparently nor

mal. The frequency of aberrant expression of these MMR genesappears to be consistent with the frequency of mutations in thesegenes observed in HNPCC (17). Although there is little informationon MMR gene mutation in human gliomas, several lines of evidencesupport the involvement of MMR genes in human gliomas. MIN ismore frequent in high-grade gliomas than in low-grade gliomas (2),suggesting that defects in MMR may be involved in glioma progression. In patients with Turcot's syndrome, a disease in which primarytumors of the central nervous system occur with colorectal polyposis(25), two distinct types of germ-line mutations have been identified:APC mutations in patients with medulloblastoma; and hMLHJ orhPMS2 mutations in patients with glioblastoma (26).

Mutations can cause premature translation termination that mediates degradation of mutant mRNAs (27, 28). However, studies haveshown that hypermethylation may be another mechanism of genesilencing in gliomas. An example is the abnormal hypermethylation of

the CpG islands in the p16/CDKN2 gene, which inactivates geneexpression by transcriptional silencing without detectable mutationsin human cancers (29), including human gliomas (30, 31). Therefore,hypermethylation of MMR genes in human gliomas may reduce

mRNA levels, as observed in the multiplex RT-PCR assay. It is alsolikely that inactivation of transcription factors may reduce the expression of the MMR genes, which may be gradually shut down as a tumorprogresses. However, the exact mechanism for the observed reducedMMRgeneexpressionin humangliomasremainsunknownandwarrants further investigation.

The relatively high frequency of reduced MMR gene expression wefound by multiplex RT-PCR in human gliomas may correlate with thefrequency of hypermethylation of promoter regions, MIN, and possi

bly MMR gene mutations. Thus, these molecular characteristics needto be correlated in a larger number of samples. This will help us betterunderstand the role of MMR genes (32) in human glioma progression.

Although a normal level of MMR protein expression has been alsoobserved in MIN-positive HNPCC tumors (33), the observed frequency (one of five) of MIN found in human gliomas with reduced

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1997;57:1673-1677. Cancer Res   Qingyi Wei, Melissa L. Bondy, Li Mao, et al.   Human GliomasMultiplex Reverse Transcription-Polymerase Chain Reaction in Reduced Expression of Mismatch Repair Genes Measured by

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