©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A hPMS2 Mutant Cell Line Is Defective in Strand-specific Mismatch Repair (*)

(Received for publication, April 24, 1995; and in revised form, June 7, 1995)

John I. Risinger (1) Asad Umar (2) J. Carl Barrett (1) Thomas A. Kunkel (2)(§)

From the  (1)Laboratory of Molecular Carcinogenesis and (2)Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and (3)Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human cells contain several homologs of the bacterial mutL gene required for mismatch repair, including a gene on chromosome 7 designated hPMS2. We have identified an endometrial carcinoma cell line, HEC-1-A, that has a C T mutation in hPMS2 that generates a nonsense codon and yields a protein truncated at the C terminus. No wild-type gene or gene product was detected. The missing amino acids in hPMS2 are highly conserved among PMS homologs, suggesting that they may be critical for function. In support of this, extracts of HEC-1-A cells are defective in repairing a variety of mismatched substrates. Moreover, di-, tri-, and tetranucleotide repeated sequences are highly unstable in single cell clones of HEC-1-A cells, and HEC-1-A cells are resistant to killing by N-methyl-N`-nitro-N-nitrosoguanidine. The results provide strong experimental support for the involvement of the hPMS2 gene product in mismatch repair in human cells and support the concept that a defective hPMS2 gene may lead to predisposition to certain forms of cancer.


INTRODUCTION

Instability of simple repetitive sequences is common in tumor DNAs from hereditary nonpolyposis colorectal carcinoma (HNPCC) (^1)kindreds and from individuals with sporadic cancers that are commonly associated with HNPCC(1, 2, 3, 4, 5, 6) . The majority of HNPCC kindreds have germline alterations in human homologs of the bacterial mismatch repair genes mutS and mutL(7, 8, 9, 10, 11, 12, 13) . Inactivation of the human hMSH2 and hMLH1 genes in cells of these individuals is thought to result in a mutator phenotype that is required for subsequent tumor progression (for review, see Refs. 14 and 15).

Supporting a role for the hMLH1 and hMSH2 gene products in mismatch repair is the finding that extracts of colon and endometrial tumor cell lines with a mutation in hMSH2 or hMLH1 are defective in mismatch repair(16, 17) . These cells also exhibit microsatellite instability, and several such lines have elevated mutation rates in endogenous genes ((16, 17, 18, 19, 20, 21) and references therein). Cell lines defective in mismatch binding and/or repair have been shown to be resistant to killing by treatment with otherwise lethal doses of DNA alkylating agents ( (20, 21, 22) and reviewed in (23) ). Moreover, the introduction of chromosome 3 containing a wild-type copy of the hMLH1 gene to a hMLH1-defective line restores DNA mismatch repair activity, reduces microsatellite instability, and increases sensitivity to MNNG (21) . These data strongly imply that both hMSH2 and hMLH1 function in mismatch repair.

Eukaryotic cells contain several genes that share sequence homology with the bacterial mutL gene. In yeast, these are designated yMLH1(24) and yPMS1(25, 26) . Interaction of the yMLH1 and yPMS1 proteins has been demonstrated by protein affinity chromatography(27) . Moreover, ypms1 mutants have elevated mutation rates and are epistatic for this phenotype with yeast ymlh1 mutants(24) , supporting a role for yPMS1 in mismatch repair. Additional genes exist in yeast that, when mutated, yield phenotypes similar to ypms1 mutants ( (28) and references therein). Human cells likewise contain several mutL homologs. In addition to the hMLH1 gene on chromosome 3(2, 8) , there are mutL homologs designated hPMS1 located on chromosome 2 (10) and hPMS2 located on chromosome 7p22(10, 29) . The latter is closely related by sequence homology to the yPMS1 gene. The hPMS2 has been described as a member of a subfamily that includes at least two related genes located on chromosome 7q(29) . Recently, Horii et al.(30) have isolated additional PMS-like sequences and have suggested that hPMS genes constitute a family containing at least 11 members.

Given the large number of hPMS sequences, what is the evidence that any of these function in mismatch repair? A role for both hPMS1 and hPMS2 in cancer, and by extrapolation, in mismatch repair, has been inferred from a mutation found in a single HNPCC family for each gene(29) . However, since HNPCC individuals are at risk for developing alterations in many genes, it is possible that these single examples of hPMS1 and hPMS2 mutations are not causally related to disease progression or to the presumed mismatch repair defect. A functional role for hPMS2 in mismatch repair is also suggested by the observation that a mismatch repair-defective extract made from a cell line containing a mutant hMLH1 gene is complemented by a purified fraction containing hMLH1 and a second protein that is either hPMS2 or a closely related gene product(31) . As noted by the authors, the identity of this second protein is uncertain because the complementing protein is 110 kDa while the deduced amino acid sequence of the hPMS2 gene product predicts a 95,808-kDa protein and because one putative peptide sequence derived from the 110-kDa protein was not found in the predicted hPMS2 polypeptide.

Thus, the role of hPMS2 in mismatch repair and in cancer is not yet as clearly established as for hMSH2 and hMLH1, where a wealth of information is available on many mutations in HNPCC families, in tumor DNA, and in tumor cell lines. Beyond the report by Nicolaides et al.(29) , no additional HNPCC kindreds, tumor DNAs, or tumor cell lines have been reported to contain mutations of any hPMS gene. Establishing a role in mismatch repair (or a lack thereof) for hPMS genes would be facilitated by identifying and characterizing tumor cell lines containing mutations in these homologs. Toward this end, we describe here a mutation in the hPMS2 gene in a human endometrial carcinoma cell line (HEC-1-A) that yields a truncated gene product but no detectable wild-type hPMS2 protein. We then show that it has the properties predicted of a cell line defective in mismatch repair.


EXPERIMENTAL PROCEDURES

Cell Lines

Human endometrial carcinoma cell lines HEC-1-A and KLE were from the American Type Culture Collection. TK6 cells were from P. Modrich (Duke University).

Single Cell Clone Analysis of Microsatellite Instability

Single cell clones of HEC-1-A and KLE cells were made from a limiting dilution of a clonal population of cells. DNA suitable for PCR was prepared from cells representing 18-25 doublings by a 1-h digestion at 56 °C in 100 µl of 1 PCR buffer (see below) without MgCl(2) and containing 100 µg/ml proteinase K. The DNA preparation was heated at 95 °C to inactivate the proteinase K before amplification. DNAs were amplified by the polymerase chain reaction using oligonucleotides specific for several repetitive sequences. Primers were from Research Genetics (Huntsville, AL), and references can be obtained from the Johns Hopkins University genome data base (or, for vWFa, see (32) ). Each PCR (10 µl) consisted of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl(2), 200 µM of each deoxynucleotide triphosphate, 1 µM of each primer, 1 µl of DNA, and 1 unit of Amplitaq polymerase (Roche Molecular Systems, Alameda, CA). One primer was end-labeled with [-P]ATP and T4 polynucleotide kinase prior to addition to the reaction. PCR was performed for 35 cycles consisting of 20 s each at 94, 55, and 72 °C. Following amplification, the reaction was diluted 1:1 in a denaturing loading buffer of 98% formamide, 10 mM EDTA (pH 8.0), 0.02% bromphenol blue, 0.02% xylene cyanol FF and denatured at 95 °C for 3 min. One-fifth of the denatured product was subjected to electrophoresis for 3 h at 90 watts in a 6% polyacrylamide gel containing 8.3 M urea and 30% formamide. Following electrophoresis gels were covered in a single layer of mylar (Ambis Systems, San Diego, CA), dried, and exposed to x-ray film.

Analysis of hPMS2 by Protein Truncation Test and DNA Sequencing

Total RNA was prepared from cell lines using the single step procedure described by Chomczynski and Sacchi(33) . First strand cDNA was prepared as described previously (34) and used as template for amplification of hPMS2 in two fragments spanning codons 1-472 and codons 415-863, using oligonucleotides containing T7 RNA polymerase sites as described(12) . A portion of each amplified product (5 µl) served as a template for coupled transcription and translation in 10-µl reactions using the TNT system (Promega, Madison, WI) that incorporates [S]methionine to label the protein product. The reactions were diluted 1:5 in SDS loading buffer and denatured at 97 °C for 5 min, and 4 µl was loaded onto 15% SDS gels. The remainder of the PCR product DNA was gel-purified and sequenced on both strands, using Sequenase v2.0. The primers used were PMS2-2282 (TTGTTATCGATGAAAATGCTC) and PMS2-2463 (GTCCCAATCATCACCGACTTC). Upon identification of the mutation (see below), a second amplification was performed, the fragment was cloned, and clones were sequenced to confirm the mutation. The hPMS2 gene sequence was determined for codons 753-860.

Mismatch Repair Assay

Cell-free extracts and mismatched substrates were prepared as described(35) . Substrates contained the indicated mismatch and a nick in the(-)-strand at position -264, where position +1 is the first transcribed nucleotide of the LacZalpha gene. Repair reactions (25 µl) contained 30 mM Hepes; 7 mM MgCl(2); 200 µM each CTP, GTP, UTP; 4 mM ATP; 100 µM each dCTP, dATP, dGTP, dTTP; 40 mM creatine phosphate; 100 mg/ml creatine phosphokinase; 15 mM sodium phosphate (pH 7.5); 1 fmol of substrate DNA; and 50 µg of extract proteins. Reactions were incubated for 15 min at 37 °C. The substrate DNA was recovered and introduced into Escherichia coli NR9162 (mutS) via electroporation and plated to score plaques as described(34) .

MNNG Cytotoxicity

Non-confluent cell monolayers were harvested and rinsed in several changes of serum-free medium, and 10^5 cells were treated with either 0, 1, 5, or 10 µM MNNG at 37 °C in serum-free medium for 45 min. Cells were rinsed twice in growth medium and then plated on 100 mM dishes at concentrations of 10^2, 10^3, and 10^4 cells/plate. After 10 days, cell colonies were fixed with methanol and stained with Giemsa, and colonies with 50 or more cells were counted. The relative surviving fraction was expressed as a ratio of the plating efficiency in treated cultures to the survivors with no MNNG exposure.


RESULTS

HEC-1-A Cells Contain a hPMS2 Gene Mutation

We examined a variety of cell lines derived from endometrial tumors(5) , the most common HNPCC-associated non-colon tumor type, for mutations in the four genes reported to have alterations in HNPCC kindreds. Among the approaches we use to screen for mutations is a protein truncation assay (12) , in which tumor cell line RNA for a specific gene sequence is converted by RT-PCR to cDNA. This DNA is transcribed and translated in vitro with [S]methionine to label the protein products, which are then resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The procedure rapidly detects shorter protein products resulting from mutations such as premature termination codons, frameshifts, deletions, or aberrant splicing. We screened a panel of endometrial tumors and tumor cell lines for mutations using primers designed to yield PCR products spanning either codons 1-472 or codons 415-862, respectively, of the hPMS2 gene(29) . The product of the latter fragment that was amplified from normal endometrial tissue generated the expected protein of 50 kDa (Fig. 1a, lane1). Although this same result was obtained with several tumor cell lines (including KLE cells, see below), we found one cell line, designated HEC-1-A, that yielded a hPMS2 polypeptide that was slightly shorter than wild type (Fig. 1a, lane2). No product of wild-type size was observed, suggesting a reduction to homozygosity for this alteration at the level of RNA expression. Parallel analyses of the other half of the gene and of the hMSH2, hMLH1, and hPMS1 genes revealed no detectable truncations of any of these genes in HEC-1-A cells (data not shown).


Figure 1: Mutation detection in HEC-1-A cells. a, protein truncation test of RT-PCR products of codons 415-862 of hPMS2. The proteins produced by in vitro transcription and translation were separated on a 15% SDS-polyacrylamide gel. Sizes of prestained molecular weight standards are indicated to the left. Lane1 is the protein produced from normal uterine tissue. Lane2 is the protein from HEC-1-A cells. b, sequence of RT-PCR products. Sequence 1 is from HEC-1-A cells, demonstrating the C to T change in the first position of codon 802 of hPMS2 (marked by an asterisk). Sequence 2 is from the normal human uterus RT-PCR product and is the wild-type hPMS2 sequence.



To determine the exact nature of the mutation in hPMS2, the sequence of the C-terminal end of the gene was determined from the PCR product that was used to generate the truncated protein. A single C to T mutation was found at nucleotide 2428 in codon 802 (Fig. 1b), creating a TGA termination codon that predicts a protein truncated by 61 amino acids. This result is consistent with the mobility shift seen in Fig. 1. No band indicative of the wild-type nucleotide was observed at this position on the sequencing gel. To confirm the absence of a wild-type gene sequence, an independent RT-PCR amplification was performed, and the resulting fragment was cloned and sequenced. Ten of 10 clones contained the expected C to T transition, and no wild-type clone was observed. To establish that the observed mutation was in hPMS2 and not in a closely related PMS gene, we sequenced 325 nucleotides of the gene, corresponding to amino acid residues 753-861. Except for the substitution at codon 802, the sequence was identical to the published hPMS2 gene sequence(29) .

Microsatellite Instability in HEC-1-A Cells

We next examined HEC-1-A cells for the microsatellite instability characteristic of tumor cell lines having mutations in the hMSH2 and hMLH1 genes(16, 17) . Single cell clones were generated from clonal populations of HEC-1-A cells and from KLE, an endometrial carcinoma cell line that had no detectable hPMS2 mutation (see above) and was proficient in mismatch repair (see below). When the clones of each line reached 18-25 population doublings, DNA was prepared and analyzed for variations at several microsatellite sequences using the PCR. Microsatellites were highly unstable in HEC-1-A clones. An example of (CTT) repeat (D7s1794) instability is shown in Fig. 2a. Eighteen of 40 single cell clones exhibited new bands, representing both expansions and contractions. Instability was noted in 3 of 40 clones for D2s147 (CA), 3 of 40 clones for D14s76 (CA), 17 of 40 clones for D14s73 (CA), and 2 of 40 for vWFa (TCTA) (data not shown). Thus, although the degree of fluctuation varied depending on the marker used, all microsatellites examined had at least one example of an altered allele. As expected of a mismatch repair-proficient cell line, no variations in microsatellite sequences were present in 29 KLE clones examined (see Fig. 2b for D7s1794, data not shown for D2s147, D14s76, and D14s73, vWA not examined).


Figure 2: Microsatellite analysis at a CTT repeat. a, analysis of single cell HEC-1-A clones. The lane marked C represents the clonal population of cells from which the single cell clones were generated. b, analysis of single cell KLE clones.



Analysis of Mismatch Repair

To determine if HEC-1-A cells are deficient in mismatch repair, cell-free extracts were prepared and tested for their ability to repair a variety of mispaired substrates in vitro. A circular M13mp2 DNA substrate was used, containing a covalently closed (+)-strand and a(-)-strand with a nick (to direct repair to this strand) located several hundred base pairs away from the mispair located in the lacZ alpha-complementation coding sequence. The (+)-strand encodes one plaque phenotype (either colorless or blue) while the(-)-strand encodes the other plaque phenotype. If the unrepaired heteroduplex is introduced into an E. coli strain deficient in methyl-directed heteroduplex repair, plaques will have a mixed plaque phenotype on selective plates, due to expression of both strands of the heteroduplex. However, repair occurring during incubation of the substrate in a repair-proficient human cell extract will reduce the percentage of mixed plaques and increase the ratio of the (+)-strand phenotype relative to that of the(-)-strand phenotype, because the nick directs repair to the(-)-strand.

As previously reported(17) , repair of substrates containing single base mismatches and one or more unpaired nucleotides is readily detected in a TK6 cell extract used as a positive control (Fig. 3). An extract of KLE cells also repairs these same mismatches. In contrast, an extract of HEC-1-A cells is defective in repair of all substrates examined. This deficiency is observed for substrates containing the nick on either the 3` or 5` side of the mismatch (Fig. 3).


Figure 3: Mismatch repair activity in three cell lines. The analysis was performed as described under ``Experimental Procedures'' using the substrates indicated (described in (17) ) and reactions incubated for 15 min. Substrates designated A contain a nick 3` to the listed mismatch, while the one designated B contains a nick 5` to the GbulletG mismatch at position 89. Results are expressed as percent repair determined from counting several hundred plaques per variable. Measurements with two of the substrates were performed multiple times with similar results. Repair values of leq1% are represented as 1%. Repair of the (Abullet-) substrate in a KLE extract was not examined. All three extracts were competent for SV40 origin-dependent DNA replication activity (e.g. see (17) ), demonstrating that the repair-defective extracts are not defective in all DNA transactions.



MNNG Cytotoxicity

Several mismatch repair-deficient cell lines have been shown to be resistant to the cytotoxic effect of treatment with MNNG ( (20, 21, 22) and reviewed in (23) ). In one instance, sensitivity to MNNG has been restored to a mismatch repair-defective, alkylation-resistant hMLH1 mutant cell line via transfer of a wild-type chromosome 3 that restores mismatch repair activity(21) . To determine if the correlation between defective mismatch repair and MNNG resistance holds for HEC-1-A cells containing a hPMS2 mutation, we treated these cells with increasing concentrations of MNNG (Fig. 4). MNNG concentrations up to 10 µM were without significant effect, while mismatch repair-proficient KLE cells were more sensitive.


Figure 4: MNNG cytotoxicity in endometrial cancer cell lines. Relative survival of HEC-1-A and KLE cells after a 45-min exposure to an increasing concentration of MNNG is shown. Each point is the average of three independent experiments.




DISCUSSION

A defective hPMS2 gene has been implicated in the development of colon cancer by the observation of a mutation in one HNPCC kindred(29) . A functional role for hPMS2 in mismatch repair has previously been suggested by the close gene sequence homology to the yeast mutator gene yPMS1(29) and by complementation of a mismatch repair-defective extract by two proteins, one of which is either hPMS2 or a closely related gene product(31) . The present study directly supports a role for hPMS2 in mismatch repair by evaluating predicted phenotypes in an endometrial carcinoma cell line containing a nonsense codon in the hPMS2 gene. Although we cannot exclude the presence of mutations in other genes in the HEC-1-A cell line, the protein truncation and DNA sequencing data (Fig. 1) establish that HEC-1-A cells do lack wild-type hPMS2. The protein that is observed is shorter and presumably missing 61 C-terminal amino acids. These amino acids are highly conserved among yPMS1, hPMS1, and hPMS2 genes(29) , suggesting that they are functionally important. The evident lack of a wild-type transcript also suggests that both copies of this gene must be inactivated in order for the defect to be manifest. This reduction to homozygosity is consistent with similar allelic inactivation observed with hMLH1 and hMSH2 mutations in cell lines and primary tumors(8, 9, 10, 12, 16, 17) . In the previously reported hPMS2 mutation, deletions were identified in both alleles of an unspecified tumor arising in this kindred, one of which was germline and the other somatic(29) .

Supporting a functional role for hPMS2 in mismatch repair is the observation that HEC-1-A cell extracts are deficient in mismatch repair (Fig. 3). The substrates used here already contain an incision to direct repair to the minus strand, and repair is defective regardless of whether the nick is 3` or 5` to the mismatch. Thus the defect is not in incision activity, but it could be prior to incision or it may involve the excision step. Both possibilities are consistent with the recent study suggesting that hPMS2 or a related protein forms a heterodimer with hMLH1(31) . This complex has been designated hMutLalpha, by analogy to the homodimeric MutL complex of E. coli, whose function is suggested to be formation of a complex with MutS that activates incision by MutH and then initiates excision of nucleotides (for review, see (36) ).

HEC-1-A cells also exhibit the microsatellite instability characteristic of mismatch repair-deficient cell lines. Instability was noted using five different repeat sequences. The greatest instability was seen with a (CTT) repeat (D7s1794, Fig. 3) and one of three (CA) repeats examined (D14s73). Instability was also detected with a (TCTA) repeat (vWFa) and two other (CA) repeats. The degree of instability was less with these latter markers, just as has been seen in other mismatch repair-defective cell lines. (^2)The instability reported here is in accord with previous reports of microsatellite instability in mismatch repair-defective hMSH2 and hMLH1 mutant cell lines(16, 17) .

As has been noted previously, mismatch repair in bacteria and humans shares a number of common mechanistic features (reviewed in (36) ). Nonetheless, E. coli contains one mutL gene while yeast and humans clearly contain two or more genes sharing close sequence homology to mutL. These homologs could be involved in substrate- or tissue-specific mismatch repair processes. The results presented in this study directly implicate a biochemical role for one of these homologs, hPMS2, in the repair of mispaired and unpaired nucleotides. They also substantiate a role for hPMS2 inactivation in the development of tumors of the endometrium, colon, and other tissues associated with HNPCC kindreds.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 919-541-2644; Fax: 919-541-7613; kunkel{at}niehs.nih.gov.

^1
The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal carcinoma; MNNG, N-methyl-N`-nitro-N-nitrosoguanidine; PCR, polymerase chain reaction; RT, reverse transcription; vWFa, von Willebrand factor a.

^2
J. I. Risinger, unpublished observations.


ACKNOWLEDGEMENTS

We thank John D. Roberts and Kenneth R. Tindall for evaluating the manuscript.


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