Human Chromosome 3 Corrects Mismatch

[CANCER RESEARCH 54, 4308-4312. August 15, 1994] Advances in Brief

Human Chromosome 3 Corrects Mismatch Repair Deficiency and Microsatellite Instability and Reduces 7V-Methyl-Ar'-nitro-Ar-nitrosoguanidineTolerance in Colon Tumor Cells with Homozygous hMLHl Mutation1

Minoru Koi, Asad t niai, Dharam P. Chauhan, Sajeev P. Cherian, John M. Carethers, Thomas A. Kunkel, and C. Richard Boland2

Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0586, and the Gastroenterology Section, VA Medical Center, Ann Arbor, Michigan 48105-2399 Â¡M.K., D. P. C., S. P. C., J. M. C., C. R. B.j, and Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Â¡A.U., T. A. K.Â¡

Abstract sporadic colon (9, 10) and endometrial (10) carcinoma cell lines. Microsatellite instability is also a hallmark of yeast with defective The human colon tumor cell line HCT 116 is known to have a homozy- mismatch repair (11). Human homologues of the yeast mismatch gous mutation in the mismatch repair gene hMLHl on human chromo repair genes hMSH2 (12, 13) and hMLHl (14, 15), have been shown some 3, to exhibit microsatellite instability, and to be defective in mis to be mutated in HNPCC kindreds. It has been hypothesized that match repair. In order to determine whether the introduction of a normal copy of hMLHl gene restores mismatch repair activity and corrects HNPCC patients lose their mismatch repair due to a germline muta microsatellite instability, a single human chromosome 3 from normal tion in one alÃelecombined with an acquired somatic mutation in the fibroblasts was transferred to HCT 116 cells via microcell fusion. As a other alÃele(9, 12). This suggests that microsatellite instability is control, human chromosome 2 was also transferred to HCT 116 cells. Two recessive and mutations in both alÃelesof either hMSH2 or hMLHl are HCT 116 microcell hybrid clones that received a single copy of chromo necessary for the manifestation of this trait. Moreover, a human some 2 (HCT 116+ch2) and two that received a single copy of chromo lymphoblastoid cell line MT1, known to be defective in mismatch some 3 (HCT 116+ch3) were isolated and characterized. A G-G mismatch repair (16), has been shown to have microsatellite instability (10), in M13-derived heteroduplex DNA was efficiently repaired in cell extracts while its parent cell line which is mismatch repair proficient shows no from HCT 116+ch3 cells, but not in those of parent HCT 116 cells or HCT microsatellite instability. The Chinese hamster ovary cell line CHO 116+ch2 cells. Microsatellite alterations at the D5SI07 locus containing MT+clone B, Raji F12, and MT1 are all defective in some component CA repeats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 150 subclones from HCT 116+ch2 cells. In contrast, none of the 225 of mismatch repair (16, 17) and show a high level of tolerance to the subclones derived from mismatch repair-proficient HCT 116+ch3 cells cytotoxic action of DNA-alkylating agents (16, 17). This has led to the showed alterations in the microsatellite at the same locus. The effect of suggestion that cell death promoted by DNA-alkylating agents may be introducing chromosome 3 on the sensitivity of HCT 116 cells to N- an active process and that the mismatch repair system may be a methyl-V-nitro-A-nitrosoguanidine (MNNG) was examined, since en general sensor for genetic damage (16). hanced tolerance to MNNG is accompanied by loss of mismatch repair In order to understand the mechanism responsible for the expres activity in several cell lines. Within 3 days after treatment with 5 Â¿IM sion of the two traits associated with mismatch repair deficiency, we MNNG, HCT 116+ch3 cells became morphologically flat and stopped transferred a normal copy of hMLHl on chromosome 3 via microcell growing. Their colony-forming ability, determined 10 days after treat fusion into the colon cancer cell line HCT 116, which has homozy- ment, was reduced 200-fold when compared to MNNG-treated parental gous mutations at the hMLHl locus (15), and is defective for mis HCT 116 and HCT 116+ch2 cells. These results support the hypothesis that mutations in both alÃelesof the hMLHl gene are necessary for the match repair activity (10). In comparison to parent HCT 116 cells, manifestation of defective mismatch repair and microsatellite instability chromosome 3-transferred HCT 116 cells have increased sensitivity to and for enhanced MNNG tolerance. The results also suggest that the MNNG, improved mismatch repair, and increased microsatellite mismatch repair system contributes to the process that causes growth stability. arrest in response to DNA damage by alkylating agents. Materials and Methods

Introduction Cell Cultures. The human colon cancer cell line HCT 116 was obtained Tumors from patients with HNPCC3 have been found to have high from the American Type Culture Collection (Rockville, MD) and maintained in Iscove's modified Dulbecco's medium containing 10% FBS. Human mono- mutation rates in microsatellite sequences (1, 2). This microsatellite chromosomal hybrids, GM11713 containing human chromosome 2 and instability has also been found in sporadic cancers of the colon (3, 4), GM11686 containing human chromosome 3 (18), were obtained from the stomach (5-7), pancreas (5), and endometrium (8). Recently, micro- National Institute of General Medical Sciences Human Genetic Mutant Cell satellite instability has been linked to a defect in mismatch repair in Repository (Camden, NJ). Both chromosomes are tagged with pSV2 neo plasmid DNA. These cell lines were maintained in DMEM containing 10% Received 6/24/94; accepted 7/12/94. FBS and 400 ng/ml of G418 (GIBCO BRL, Gaithersburg, MD). The costs of publication of this article were defrayed in part by the payment of page Microcell Transfer. The methods for microcell chromosome transfer were charges. This article must therefore be hereby marked advertisement in accordance with similar to those described previously (19). Briefly, the monochromosomal 18 U.S.C. Section 1734 solely to indicate this fact. hybrid cells (3 X IO7) were seeded into twelve 25-cm2 flasks (Costar No. ' This work was partly supported by N1H Grant CA39233, Gastrointestinal Hormone Research Core Center P30 DK34933, the University of Michigan Cancer Center 3025; Costar, Cambridge, MA) in DMEM containing 10% FBS plus 80 /xg/ml CA46592-Ãœ6,and the Johnson Family Fund. of G418. Two days later, the medium was changed to a fresh growth medium 2 To whom requests for reprints should be addressed. â€¢'Theabbreviations used are: HNPCC, hereditary non-polyposis colon cancer; MNNG, containing 0.05 fig/ml of Colcemid (Sigma Chemical Co., St. Louis, MO) and /V-methyl-W-nitrO'/V-nitrosoguanidine; PCR. polymerase chain reaction; SSCP, single- cultures were mitotically blocked for 2 days to induce micronuclear formation strand conformation polymorphism; FBS, fetal bovine serum; DMEM, Dulbecco's mod in the cells. The flasks were filled with prewarmed, serum-free DMEM ified essential medium. containing 10 fig/ml cytochalasin B (Sigma) and securely placed into Dupont 4308

GSA rotor wells filled with 140 ml warm (30Â°C) water. The flasks were creatine phosphokinase; 15 mM sodium phosphate (pH 7.5); 1 fmol of the centrifuged at 1000 rpm for 1 min for balancing, followed by centrifugation at indicated heteroduplex DNA; and 50 /ig of cell extract protein. After incubat K).O(K) x g for l h at 30Â°C.The microcells were resuspended in 10 ml of ing at 37Â°Cfor 30 min, reactions were processed and introduced into Es- serum-free DMEM and filtered in series through 8-, 5-, and 3-jj.m polycar cherichia coli NR9162 (mulS), which were plated to score plaque colors (26). bonate filters (Nuclcpore, Pleasanton, CA). The purified microcells were Repair efficiency is expressed in percent as 100 X (1 - the ratio of the pelleted by centrifugation at 500 x g for 7 min and resuspended in 2 ml of percentages of mixed bursts obtained from extract-treated and untreated serum-free medium containing 100 fig/ml phytohemagglutinin (Sigma). The samples) (27). microcells were attached to recipient cell monolayers by incubating for 15 min Assay for Microsatellite Instability. Single cell clones were isolated from at 37Â°C.The cultures were treated with 3 ml of 47% polyethylene glycol (M, 10-cm-diameter culture plates containing about 100 separated colonies by 8000; Sigma) for 1 min followed by extensive washing in serum-free medium. sampling each colony with micropipets. The cells were inoculated into 96- After 24 h, the cells were trypsinized and split into 5 plates (5 cm diameter) multiwell plates and grown to confluency. The plates were washed twice with containing selective medium with 400 jj.g/ml G418. The resulting G418- phosphate-buffered saline, and 50 jul containing 100 ^ig/ml of proteinase K in resistant microcell hybrids were isolated after 10 to 14 days of growth. 10 mM Tris (pH 8.0) and 1 mM EDTA were added to each well. The plates were Chromosomal Analysis. Cells were prepared for chromosome analysis by incubated first for 2 h at 65Â°Cand then for 15 min at 95Â°C.One /il of each the methods described previously (19). Chromosomes were banded with quin- DNA sample was used for each PCR reaction. The gene locus used to detect acrine mustard (20). Ten well-banded metaphase spreads were karyotyped. microsatellite instability was D5S107, using primers 5'-GATCCACTTTAAC- Southern Blot Hybridization. Genomic DNA was isolated by the method CCAAATAC-3' and 5'-GGCATCAACTTGAACAGCAT-3' (28). The PCR of Miller et al. (21). DNA was digested with the Msp\ (Boehringer Mannheim, conditions were the same as those used for the hMLHl gene described above. Indianapolis, IN) restriction endonuclease. Digested DNA was analyzed by After PCR amplification, the products were denatured and analyzed on a electrophoresis in 1% agarose gel and transferred to nitrocellulose filters in sequencing gel of 8% polyacrylamide with 7.5 M urea. alkaline conditions (22). A radioactively labeled probe was prepared by the MNNG Cytotoxicity Test. Exponentially growing cells (10s) were sus method of Feinberg and Vogelstein (23), and hybridization was carried out by pended in 1 ml of serum free RPMI 1640 and treated with various concentra the method of Church and Gilbert (22). The probe used, pYNH24(D2S44) (24), tions (0, 1.25, 2.5, and 5 /IM) of MNNG for 45 min at 37Â°C.After treatment, was obtained from the American Type Culture Collection. the cells were washed once with serum-free medium; resuspended in fresh SSCP-PCR oÃhMLHl. A DNA segment encompassing codons 228 to 263 growth medium: diluted; and plated into duplicate wells at 10J, 10\ IO2, 10, of the hMLHl gene was amplified using a sense primer, 5'-TGATAGAAAT- and 1 cell/well. Ten days later, the cells were fixed with methanol and stained TGGATGTGAGG-3', and an antisense primer, 5'-TGATGAAGAGTAA- with hematoxylin, and the number of colonies consisting of more than 50 cells GAAGATGC-3' (15). The sense primer was end labeled with [7-32P]ATP were counted. The relative surviving fraction for each cell line was expressed (6000 Ci/mmol; Amersham, Arlington Heights, IL). The PCR was performed as a ratio of the plating efficiency in treated cultures to that observed in the in 5-fil volumes of a mixture containing 1 X PCR buffer [10 mM Tris (pH controls. 8.3)-50 mM KC1-1.5 mM MgCl2-0.001% gelatin], 10 pmol each of unlabeled primer and labeled primer, 20 ng template DNA, 0.25 unit Taq DNA polym- Results and Discussion erase, and 250 fiM concentrations of each dideoxynucleotide. The reaction tubes were heated to 94Â°Cfor 2.5 min and then cycled 35 times; each cycle A total of 60 independent G418-resistant colonies were formed consisted of 1 min at 93Â°C,1 min at 55Â°C,1 rain at 72Â°C,and 10 min at 72Â°C after three successive chromosome 3 transfer experiments. Similarly, for final elongation in a thermal cycler (Perkin Elmer Cetus, Emeryville, CA). 49 independent G418-resistant hybrid clones were obtained after SSCP analysis of PCR products was performed by the method described chromosome 2 transfer experiments. Two clones from each were previously (25). PCR products were denatured and separated on a 0.5 X Hy- further analyzed for the intact transfer of single copies of chromosome droLink MDE Gel (J. T. Baker Chemical Co., Phillipsburg, NJ) containing 5% 2 or 3. Karyotypic analysis with quinacrine mustard banding revealed glycerol, 0.054 MTris-borate, and 0.0012 M EDTA at 4Â°C.After electrophore that the parental HCT 116 line contained two intact chromosomes 2 sis, the gel was dried and exposed to X-ray film for 12-16 h. and two intact chromosomes 3, whereas two microcell hybrid clones Mismatch Repair Assay. Cell free extracts were prepared as described obtained from chromosome 2 transfer experiments contained one (26). Procedures for mismatch repair have been described (26, 27). Mis additional chromosome 2. Southern blot analysis of the D2S44 locus matched substrates, prepared as described (27), contained a nick in the minus strand at position â€”264,where position +1 is the first transcribed base of the also detected a new fragment in the hybrid clones, the size of which lacZat gene. Repair reactions (25 JA!)contained 30 mM 4-(2-hydroxyethyl)-l- was the same as that of chromosome 2 donor GM11713 cells (Fig. piperazineethanesulfonic acid (pH 7.8); 7 mM MgCU; 4 mM ATP; 200 JU.M LA), thus confirming the successful transfer of chromosome 2. Band concentrations each of CTP, GTP, and UTP; 100 JIM concentrations each of ing analysis also showed the presence of an additional copy of dATP, dGTP, dTTP, and dCTP; 40 mM creatine phosphate; 100 mg/ml of chromosome 3 in the two microcell hybrid clones obtained from

N <Ã² <0

Fig 1. Presence of transferred chromosome 2 or 3 // in recipient HCT 116 cells. A, Southern blot analysis A. VÂ¿r V^ ^V B- of DNA from chromosome 2 donor GM11713 cells, parental HCT 116 cells, and two HCT 116 +ch2 microcell hybrids, clone 1 and clone 3. In two HCT & & / ^ ^ 116+ch2 clones, both donor and recipient D2S44 fragmenls were detected (arrows). B, detection by SSCP-PCR of the donor hMLHl gene in two HCT116 +ch3 microcell hybrids, clone 5 and clone 6. In two HCT 116+ch3 clones, both donor and recipient bands were detected (arrows).

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Table 1 Restoration of mismatch repair activity by chromosome 3 transfer" subclones from HCT 116 clone A were examined at the D5S107 (%)Blue48494226White15172062Totalpfu*370483437662% microsatellite locus, 8 clones (10%) showed band shifts in the ampli- of repairc09068B/W3.32.82.10.4cons (Table 2), representing both deletions and elongations of the CellextractMockHCT repeats (Fig. 2A), confirming previously reported (9) microsatellite

AHCT116 clone instability in the parental cell line. The same analysis for the HCT 116 -t-chX1clone 116+ch2 clone 1 (Fig. 2B) and clone 3 showed that 6 of 82 (7.3%) 3HCT 116 +ch3''clone and 7 of 68 (10.3%) of the subclones, respectively, exhibited band 6PlaqueMixed37343812phenotype shifts (Table 2). In contrast, 0 of 127 and 0 of 119 subclones from "A heteroduplex containing a G-G mispair at position 88, a blue (-) strand, a HCT 116+ch3 clone 5 and clone 6 (Fig. 2C) demonstrated mutations white (+) strand, and a 3' nick al position -264 in the - strand was used for repair (Table 2). These results indicate that chromosome 3, but not chromo reactions (10). 1pfu, plaque-forming units; B/W, ratio of blue plaques to white plaques. some 2, corrected microsatellite instability in HCT 116 cells. ' Repair efficiency was expressed in percentages 100 X (1 â€”ratio of the percentages Next, parental HCT 116 cells, HCT116+ch2 cells, and HCT of mixed bursts obtained from extract-treated and mock treated samples). 116+ch3 cells were tested for sensitivity to the cytotoxic effects of HCT 116 microcell hybrid which received a single copy of chromosome 2. ' HCT 116 microcell hybrid which received a single copy of chromosome 3. MNNG. The cells were treated with increasing concentrations of MNNG for 45 min at 37Â°C,washed, and replated for colony-forming ability. Three days after treatment, most of the HCT 116 + ch3 cells chromosome 3 transfer experiments. Transfer of the donor hMLHl treated with 5 /XMMNNG became morphologically flat and their locus to these two clones was examined by SSCP analysis of PCR growth was arrested. During further cultivation, these cells accumu products from DNA segments encompassing codons 228 to 263 of lated a large number of cytoplasmic vacuoles and eventually detached hMLHl. Since recipient HCT 116 cells contain homozygous muta from the plates. In contrast, the same dose of MNNG had little effect tions at codon 252 oÃhMLHl, it is possible to separate the mutated on either the morphology or growth of parental HCT 116 cells and recipient and normal donor PCR segments on a SSCP gel. As shown HCT 116+ch2 cells. Ten days after treatment, the cells were fixed in Fig. IÃŸ,HCT 116+ch3 clone 5 and clone 6 contained an additional and stained and the relative fraction of the surviving colonies was band from the chromosome 3 donor GM11686 cells. Based on these determined. As shown in Fig. 3, the introduction of chromosome 3 results, these hybrid clones were used for further experiments. reduced tolerance to MNNG cytotoxicity 30-fold at 2.5 /AM and First, mismatch repair activities in cell extracts from HCT 116 200-fold at 5 JAMas compared with the tolerance observed in the clone A (a clonal derivative from HCT 116), HCT 116+ch2 clone 3, parental cells. The introduction of chromosome 2 had no effect on and HCT 116+ch3 clone 6 were measured. To measure the ability of sensitivity to MNNG cytotoxicity. these extracts to repair mismatches, we used an M13 DNA substrate As shown in this report, introduction of a single copy of chromo containing a covalently closed ( + ) strand and a (-) strand with a nick some 3 into HCT 116 cells resulted in a correction of mismatch repair located several hundred base pairs away from an unpaired nucleotide deficiency and microsatellite instability. Our results strongly suggest in the /acZa-complementation coding sequence. The two strands that the hMLHl product from one copy of a normal hMLHl gene is encode different plaque colors, i.e., blue versus colorless (or white). If sufficient to restore mismatch repair activity and microsatellite sta an unrepaired heteroduplex is introduced into an E. coli strain defi bility. Moreover, the transfer of a single intact chromosome ensures cient in methyl-directed heteroduplex repair, the plaques will have a that the encoded genes are likely to be expressed at normal levels in mixed phenotype on selective plates, due to expression of both strands the recipient cells. This supports the hypothesis that inactivation of of the heteroduplex. However, repair occurring during incubation of both copies of hMLHl, leading to complete loss of mismatch repair the substrate with a repair-proficient human cell extract will reduce activity, is necessary for the manifestation of microsatellite instability. the percentage of mixed plaques and increase the ratio of the (+) The cell lines that lack mismatch repair activity are resistant to strand phenotype relative to that of the (-) strand phenotype, since the relatively high concentrations of MNNG (16, 17). In this study, we nick directs repair to the ( â€”)strand (27, 29). The introduction of a found that tolerance to MNNG is significantly reduced in the mis M13 heteroduplex containing a G-G mispair directly into an E. coli match repair-proficient HCT 116+ch3 cells. These results indicate mutS strain without prior incubation in a cell extract yielded 37% that the mismatch repair system is involved in mediating MNNG mixed color plaques (Table 1). Incubation of this substrate in extracts cytotoxicity. Other studies have shown that MNNG treatment causes of either HCT 116 clone A cells or HCT 116+ch2 clone 3 cells did growth arrest and immediate, apoptosis-like cell death and that the not reduce the percentage of mixed plaques, indicating that these proportion of dying cells is MNNG dose-dependent (30, 31). In extracts were deficient in repair of the G-G mismatch. However, this contrast, HCT 116 cells which received one copy of chromosome 3 same substrate was efficiently repaired in HeLa cell extracts (10) and did not show immediate cell death at any concentration of MNNG up in an extract of HCT 116+ch3 clone 6 cells (Table 1). Strand-specific repair was indicated both by the reduced percentage of mixed plaques and by the selective decrease in the proportion of blue plaques the Table 2 Correction of microsalellitc instability (MSI) by chromosome 3 transfer nicked (-) strand phenotype with a concomitant increase in the of clones with MSI/no, of clones proportion of colorless (or white) plaques the ( + ) strand phenotype CelllineHCT examined(%)"8/80 (Table 1). Heteroduplex repair was examined a second time, but using AHCT116 clone (10)6/82 a heteroduplex containing two unpaired bases rather than a mispair. 116 + ch2 Again, the extract of HCT 116+ch3 clone 6 cells repaired the het Clone 1 (7.3) eroduplex in a strand-specific manner (71% repair in 30 min) while Clone3HCT 7/68(10.3)0/127 NS>0.001 extracts of either HCT 116 clone A or HCT 116+ch2 clone 3 cells did 116 + ch3 not. Thus, the introduction of a single chromosome 3 restored Clone 5 (0) mismatch repair activity to HCT 116 cells. Clone 6No. 0/119(0)tNS* >0.001 " Microsatellite instability was examined at the D5SI07 locus. In order to determine the effect of transferred chromosome 2 or 3 ' Statistical significance of correction of microsatellite instability was determined by on microsatellite instability in HCT 116 cells, microsatellite amplifi X~ analysis, which compared each chromosome-transferred clone to HCT 116 clone A. cation was performed on single cell clones of each cell line. When 80 c NS, not significantly different from control (HCT 116 clone A). 4310

A. HCT116cloneA B. HCT 116+ch2 clone 1 AAA

Fig 2. Microsatellite stability; alterations of CA re peats in the D5S107 locus in HCT 116 subclones. A, subclones derived from parental HCT 116 clone A. B, subclones derived from HCT 116+ch2 clone 1; C, stabilization seen in subclones derived from HCT 116+ch3 clone 6. T, deletions, A, insertions. Alter C. HCT116+ch3clone6 ations in either of the two alÃeles(arrows) are indicated above (large alÃele)or below (small alÃele)the bands.

sense the degree of damage necessary to induce cell death but may be enough to induce growth arrest. It would be interesting to know whether HCT 116 cells that receive two copies of normal hMLHl gene show both immediate cell death and growth arrest upon MNNG HCT116 treatment. HCT116 +Ch2 clone 3 Acknowledgments HCT116 +Ch2 clone 1 We would like to thank Drs. }. Carl Barrett and Giancarlo Marra for their critical reviews, Dr. Lucy M. Anderson for kindly providing the MNNG, and Karla Freiheit for typing the manuscript.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. 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 hMLH1 Mutation

Minoru Koi, Asad Umar, Dharam P. Chauhan, et al.

Cancer Res 1994;54:4308-4312.

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