Mismatch Repair in Extracts of Werner Syndrome Cell Lines

CANCER RESEARCH57. 2956-2960. July 15. 19971 Mismatch Repair in Extracts of Werner Syndrome Cell Lines

Samuel E. Bennett, Asad Umar, Junko Oshima, Raymond J. Monnat, Jr., and Thomas A. Kunkel' Laboratory of Molecular Genetics, National institute of Environmental Health Sciences, Research Triangle Park. North Carolina, 27709 i'S. E. B., A. U., T. A. K.]: and Department of Pathology, University of Washington, Seattle, Washington, 98195 Ii. 0., R. J. M.J

ABSTRACT The function(s) of the WRN protein are unknown. Several obser vations suggest that it may participate in one or more DNA transac Werner syndrome (WS) is an autosomal recessive disease, the pheno tions required for maintaining genetic stability in somatic cells. Cells type of which is a caricature of premature aging. WS cells and cell lines from WS patients exhibit multiple, clonal chromosome rearrange display several types of genetic instability, and WS patients have an ments (termed variegated translocation mosaicism; Refs. 13 and 14), increased risk of developing cancer. The WS locus (WRN) encodes a protein that shows significant sequence homology to the RecQ family of and SV4O-transformed WS fibroblast cell lines exhibit a deletion DNA helicases. Because a DNA helicase may function in DNA mismatch mutator phenotype (15). WS patients have elevated frequencies of repair, we examined extracts of WS cell lines for mismatch repair activity. HPRT-deficient T cells and of glycophorin-A variant RBCs3 (16, 17). Extracts from four different WS lymphoblastoid cell lines containing The limited in vitro replicative life span of primary WS fibroblasts different WRNmutations and from three within-pedigree control cell lines (18) and the slow growth and impaired S-phase transit of WS B- were all proficient in mismatch repair. In marked contrast, extracts from lymphoblastoid cell lines ( 19) also suggest a possible defect in DNA three independent WS fibroblastoid cell lines were deficient in repair of metabolism. base-base and insertion/deletion mismatches. Extracts ofone of these lines The increased risk of malignancy in WS patients could arise from restored activity to extracts of mismatch repair-deficient tumor cells with a DNA repair defect. Precedents for such an association exist in the defined mutations in hMSH2, hMSH3, hMSH6, hMLHJ, or !ZPMS2.This correlation between skin cancer and a defect in nucbeotide excision suggests that the WRN mutation in this fibroblast line is not a dominant negative Inhibitor of mismatch repair activity and that the repair defect repair in xeroderma pigmentosum patients (reviewed in Ref. 20). does not reside in these five known mismatch repair genes. Defective Defects in DNA mismatch repair are also linked to hereditary non mismatch repair in fibroblastoid but not lymphoblastoid cells is consistent polyposis colorectal cancer and to numerous sporadic cancers (re with the possibility that WRN protein could have a cell type- and/or viewed in Refs. 21 and 22). Studies of the link between cancer and tissue-specific role in mismatch repair. Alternatively, a mutation in WRN microsateblite instability have revealed the identities and functions of could predispose cells to mutations in other genes required for mismatch several of the genes and gene products involved in the early steps of repair activity, at least one of which could be an unknown gene. DNA mismatch repair (reviewed in Refs. 22â€”24).Similar to the way Escherichia coli requires DNA helicase II for mismatch repair (re INTRODUCTION viewed in Ref. 25), a helicase could be required for mismatch repair in human cells. The identification of the WRN protein as a potential WS2 (McKusick catalogue no. 277700) is an uncommon, autoso helicase and the causal links that have been established between loss mal recessive disease, the phenotype of which mimics premature of helicases or helicase-like proteins and genetic instability, elevated aging. As young adults, WS patients develop bilateral cataracts, cancer risk, and DNA repair deficits (26â€”28)prompted us to examine graying and loss of hair, osteoporosis, atherosclerosis, and diabetes. a possible role of the WRN protein in DNA mismatch repair in human WS patients are also at increased risk of developing cancer. The most cells. common malignant neoplasms are thyroid carcinoma, bone and soft tissue sarcomas, and malignant melanoma, and meningiomas and thyroid adenomas are predominant among the benign neoplasms MATERIALS AND METHODS identified in WS patients (reviewed in Refs. 1 and 2). Thus, the Cell Culture. The cell lines used in this study are described in Table 1.The clinical phenotype of WS shows overlap with, but is distinct from, WS SV4O-transformedfibroblastoid cell line PSV8I 1 and the WS and control normal aging in several respects (1). B-lymphoblastoid cell lines were obtained from the International Registry for The WS locus, WRN, was originally mapped to chromosomal Werner Syndrome at the University of Washington (4). WS SV4O-transformed region 8pl2 (3â€”7).The WRN gene was recently isolated (8) and fibroblastoid cell lines W-V and WS780/AG1 1395 were obtained from J. Carl encodes a predicted protein of 1432 amino acid residues (Mr 162,500). Barrett (National Institute of Environmental Health Sciences). The WRN The primary amino acid sequence contains a central region of 293 mutations in the B-lymphoblastoid cell lines and in WS780/AG113954 were amino acids that contains seven highly conserved helicase consensus determined by Oshima et a!. (7). All other cell lines have been described domains and a 570-residue NH2-terminab domain and a 569-residue previously (29, 30), except for the endometrial cell line HOUA, in which the COOH-terminal domain, both of which back homology to known hPMS2 protein is not detectable.5 WS and control B-lymphoblastoid cell lines were grown in RPMI 1640 proteins. The helicase consensus domain region of WRN shows strong supplemented with 10% fetal bovine serum (Hycbone Laboratories, Logan sequence homology with members of the bacterial RecQ family of UT). Other cell lines were grown in a 1:1 mixture of DMEM and Ham's F-12 helicases. In addition to RecQ and WRN, this family includes BLM, medium, supplemented with 10% fetal bovine serum. All growth media were the recently identified Bloom syndrome gene product (9), the Sac supplemented with 30 mg/liter penicillin G, 50 mg/liter streptomycin, and 2.5 charomyces cerevisiae SGSJ gene (10, 11), and the Caenorhabditis mg/liter FungizoneÂ© (Life Technologies, Inc.). Cultures were maintained at elegans CELFI8C5c gene (8, 12). 37Â°Cina humidified 5% CO2 incubator. Mismatch Repair and Replication Activity Assays. Preparation of cell Received 1/I 3/97; accepted 5/15/97. extracts, sources of materials, construction of substrates, and all details of The costs of publication of this article were defrayed in part by the payment of page the mismatch repair and SV4O replication activity assays have been de charges. This article must therefore be hereby marked advertisement in accordance with scribed (31, 32). Mismatch repair was investigated using heteroduplex 18 U.S.C. Section 1734 solely to indicate this fact. @ To whom requests for reprints should be addressed. Phone: (919) 541-2644; Fax: (919) 541-7613: E-mail: [email protected]. 3 R. J. Monnat, unpublished results. 2 The abbreviations used are: WS, Wemer syndrome; HPRT, hypoxanthine phospho 4 J. Oshima and G. M. Martin, unpublished results.

@@ ribosyltransferase. S Risinger. unpublished results. 2956

studyCell Table 1 Cell lines used in this

(Ref.)EBV-transformed lineMutant alleleâ€•positionâ€•Predicted proteincCommentsâ€• linesSY90575l&2:lymphoblastoidcell (8)SY90576NoneWild CAGâ€”sIAG STOP/37241 164 aaVHomozygous mutant heterozygoteZM9O630l&2: typeUnaffected sibling potential Gâ€”@C/3370--34641060 asHomozygous splice site mutation leads to (8)ZM90633NoneWild skipped exon, frameshift, and stop heterozygoteAUS40025I typeUnaffected sibling potential : A deletion/l 395 as mutant allele I (7) 2: GAâ€”sOTsplice site mutation391 I 157 aaAUS AUS mutant allele 2 (7); splice site mutation creates I l3-bp deletion encompassing cDNA (8)AUS4001O1 nts 3691â€”3803 : A deletion/l395 as father has AUS mutant allele I (7)0W90650l&2: 2: None391 Wild typeCarrier Wild and type allele (7)SV4O-transformed CGAâ€”@TGASTOP/l336368 aaHomozygous mutant linesPSV81 fibroblastoid cell 1l&2: not yet definedNot yet definedDerived from 29 yr-old female WS patient (37); exhibits chromosome instability and spontaneous deletion mutator phenotype at 38)W-V1: the HPRTlocus (15. G deletion/3004 protein; W-V mutant allele l@ 2: Not yet definedNot yet definedAltered W-V mutant allele 2; line is phenotypically similar to PSV81 I. Derived from 45 yr-old (39)WS78O/AG1 male WS patient 1395l&2: CGAâ€”@IGASTOP/l336368 asHomozygous for same mutation as OW pedigree4;derivedfrom60 yr-oldmaleWS patient (40); displays normal sensitivity to UV and DNA-damaging drugs (40) a 1 and 2 refer to the two WRN alleles contained in patient OR control cell lines. b Position refers to the position of altered nucleotides in the WRN eDNA sequence (8). Splice site mutations are detailed with an indication of their consequences (see comments). CThepredictedform of the WRN proteinwasdetenninedfromtheWRN cDNA sequenceandfrom thepresence,position,andconsequencesofopenreadingframeor splicesite mutations on the 1432-residue WRN open reading frame. d The allele status of WS and control, potential heterozygous carriers is detailed where known, as are the consequences of splice junction mutations. Cellular phenotypes of SV4O-transformed fibroblastoid cell lines are given. References refer to mutation characterization in the WS and control EBV-transformed lymphoblastoid cell lines and to cell line derivation and mutation delineation in the SV4O-transformedfibroblastoid cell lines. C @,amino acid; nt, nucleotide.

Ml3mp2 DNA containing a single base-base mismatch or two extra bases (33, 34), synthesis is not required because the gap generated by mismatch in one strand. For example, one substrate contains a G-G mismatch and excision can be filled in by an E. coli polymerase after introduction of the consists of a covalently closed (+) strand that encodes a colorless or DNA into the a-complementation host [see Fig. 1 in Umar et a!. (35)1. The â€œwhiteâ€•plaquephenotype and a nicked complementary (â€”)strand that calculation of mismatch repair efficiency is described in Thomas et al. (31). encodes a blue plaque phenotype. The nick directs repair to the (â€”)strand Details of individual experiments are given in the legends to Table 2 and and is located either 3' (GIG-A) or 5' (GIG-B) to the mismatch, which is Figs. 1â€”3. located at position 88 of the lacZa-complementation coding sequence. When introduced into an E. coli strain deficient in methyl-directed hetero RESULTS duplex repair (e.g., mutS), an unrepaired G-G heteroduplex yields a â€œmixedâ€•plaquephenotype due to expression of both strands of the Mismatch Repair in Extracts of WS B-Lymphoblastoid Cell Ml3mp2 DNA. However, if repair of the mismatched base situated on the Lines. The WS cell lines used in this study are listed in Table 1, and (â€”)strandtakes place during incubation with a human cell extract, the percentage of mixed plaques decreases and the percentage of colorless (+) the organization of the predicted WRN protein and the location of the plaques increases, such that the ratio of pure blue to pure colorless plaques various WRN mutations are shown in Fig. 1. When extracts of the is reduced. Detection of mismatch repair activity with this assay only lymphoblastoid cell lines were examined for mismatch repair, both requires excision of the mismatched base(s) in the nicked strand. Although WS and unaffected pedigree-control lines repaired a G-G mismatch, DNA repair synthesis in the extract does occur under normal circumstances with an efficiency similar to that observed with a HeLa cell extract.

Table 2 Complementation analysis with mismatch repair-defective cell extracts Complementation reactions and analysis were performed as described in â€œMaterialsand Methods.â€•The substrate contained a G-G mispair at position 88 and a 5' nick at position +276. repairâ€•ExtractPSV8I %

1 16â€• /hMSH3PSV81 WRIrW-V WR.NWS780 WR.NHEC59b hMSH2HCT15C hMSH6HHUAd hMSH3/6HOUAC PMS2HCTI hMLHI I W-V 260 14 37 52 8 4 25 WS7807.40.2 554 6075 4839 634 542 II aTherepairvalueslistedaretheaverageoftwoexperiments,exceptforthevaluesforW-VorWS780incombinationwithHHUA,whicharesingledeterminations.Repairefficiency values below 10% are in the range of experimental fluctuation in the assay and should not be considered as evidence that strand-specific repair is occurring. b The endometrial carcinoma cell line HEC59 is heterozygous for two mutations in hMSH2, one a CAAâ€”STAA mutation in exon 7 and the other a 4-base insertion in exon 9 (30). CColorectalcarcinomacell line HCT15containstwo hMSH6mutations,a l-bp deletionat codon22 (Leuâ€”s'nonsense)anda GATAGAâ€”*Tmutationat codon I 103,creatinga termination codon 9 bp downstream (41). d The endometrial carcinoma HHUA cell line is homozygous for two mutations, one a deletion of an A-T base pair at position 1 148 in the hMSH3 gene that results in a protein 723 amino acids shorter than the wild type and the other a homozygous C--sT mutation at nucleotide 3655 of the hMSH6 gene, changing codon 1219 from conserved threonine to an isoleucine (42). V The hPMS2 protein is not detectable by western blot in the HOUA endometrial carcinoma cell line.5 -â€œThecolorectalcarcinoma cell line HCTI 16 is hemizygous in hMLHI for a TCAâ€”*TAAtruncationat codon 252 in exon 9 (43) and is also homozygous for the same l-bp deletion in hMSH3 that is present in HHUA cells.5 2957

WS780 cells were used in pairwise combinations with each other. Efficient repair was observed when PSV81 1 and WS780 were included in the same reaction, but little or no repair was observed with the other two N-@ I combinations (Table 2). This suggests that the nature of the mismatch HR@ repair defects is different in these WS fibroblast cell lines. This same ZM AUS2 suggestion derived from studies in which extracts of PSV8I 1, W-V, AUS1I and WS780 cells were used in pairwise combinations with extracts of Fig. 1. Organization of the predicted WRN protein and position and type of mutations mismatch repair-deficient human tumor cell lines harboring defined identified in cell lines used in this study. The predicted WRN protein consists of a mutations in five different known mismatch repair genes (Table 2). 1432-residue open reading frame. The 570 residue N-terminal region (0, left) has no The addition of a PSV8 11 extract restored repair to reactions contain known function and contains several clusters of charged acidic residues. The 293-residue central region (U) contains seven helicase consensus domains that show strong organi ing mismatch repair-defective extracts prepared from cells mutant in zational and sequence homology with the members of the bacterial RecQ family of hMSH2 (HEC59), hMSH6 (HHUA and HCT15), hMSH3 (HHUA and helicases; the COOH-terminal region of 569 residues (0, right) includes several clusters of charged amino acid residues. The positions of chain termination mutations are indicated HCT1 16), hPMS2 (HOUA), or hMLHI (HCT1 16; Table 2). In con by open arrows above and frameshift mutations by closed arrows below the protein trast, addition of an extract of W-V cells restored repair to reactions schematic. The positions of two exon deletions generated by splice junction mutations are containing mismatch repair-defective extracts prepared from cells indicated by bracketed lines below the protein schematic. Mutation designations refer to specific WS pedigrees in the Intemational Registry for Wemer Syndrome. The WS780 mutant in hMSH2 (HEC59), /ZMSH6 (HCT15), or hMSH3 and hMLHJ cell line has been previous described (40). (HCT116), but not to extracts of HOUA cells (lacking hPMS2 pro tein) or extracts of HHUA cells mutant in hMSH3 and hMSH6 (Table 2). The results with an extract of WS780 cells were similar to those Repair was proficient, with substrates containing the nick either 3â€˜or obtained with an extract of W-V cells, except that complementation of 5' to the mismatch (Fig. 14). That repair was strand specific is an extract of HCT1 16 cells was minimal (1 1%; Table 2). In all cases indicated by the decrease in the ratio of blue to white plaques (Fig. 14, where functional complementation of repair was observed, the change inset). Proficient repair of substrates containing a 2-base loop was also in the ratio of blue to white plaques relative to the unrepaired control observed in all of the WS-afflicted and unaffected pedigree-control was consistent with strand-specific repair. cell lines. Again, repair was strand specific (Fig. 2B, inset) and was observed when the nick was either 3' or 5' to the loop (Fig. 2B). DISCUSSION Mismatch Repair in Extracts of WS Fibroblast Cell Lines. We then examined mismatch repair in extracts of the WS fibrobbastoid This study was undertaken to determine whether the WS gene cell lines. In these experiments, a (positive control) HeLa cell extract product functions in mismatch repair. Extracts of four different EBV was proficient in repair of substrates containing the G-G mismatch transformed lymphoblastoid cell lines containing characterized muta and either a 5' or a 3' nick (Fig. 3), and the blue to white plaque ratio tions in the WRN gene (Table 1 and Fig. 1) were all proficient for declined, as expected for stand-specific repair (Fig. 3, inset). How repair of a G-G mismatch (Fig. 14). This mismatch is a good substrate ever, extracts of the WS cell lines PSV81 1, W-V, and WS780 were all for the general mismatch repair system in humans that requires both defective in repair of these substrates (Fig. 3). The repair deficiency in the MutScr heterodimer of hMSH2 and hMSH6 and the MutLa the WS780 cell extract is particularly interesting because WS780 cells heterodimer of hMLH1 and hPMS2 (for review see Refs. 23 and 24). contain the same WRN mutation present in the OW lymphoblastoid The WRN-mutant lymphoblastoid cell extracts were also proficient in cell line that was repair proficient (Fig. 2). the repair of a substrate containing two unpaired bases (Fig. 2B), a Extracts of PSV8 11 were also found to be defective in repair of a process thought to be initiated by binding of a heterodimer of hMSH2 substrate containing a T-G mismatch or one, two, or four unpaired and hMSH3. Proficient repair of both base-base and insertion/deletion bases (data not shown). Each of the three WS fibrobbast extracts was substrates occurred despite the fact that the four WRN-mutant cell competent for SV4O origin-dependent DNA replication (see legend to lines contain very different WRN mutations that, in at least one case Fig. 3), demonstrating that they are not generally deficient in all DNA (OW), eliminated potential hebicase activity of the WRN protein (Fig. transactions. Moreover, no inhibition of repair of a G-G mismatch by 1). OW is homozygous for a nonsense codon in the NH2 terminus of repair-proficient HeLa cell or WS lymphoblastoid cell extracts was WRN, and the predicted protein consists of only 368 amino acids observed when these were mixed with PSV8I 1, W-V, or WS780 (Table 1). These results indicate that neither the complete WRN gene extracts (data not shown). Thus, extracts of WS fibroblastoid lines did product nor predicted helicase activity encoded by the conserved not contain a trans-acting inhibitor of repair. Extracts of two cell lines hebicase motifs plays an essential role in hMSH2/hMSH6- or hMSH2/ obtained by SV4O transformation of normal fibrobbastoid cells, 1D4 hMSH3-dependent strand-specific mismatch repair in human B-lym (36) and GM637 ( 15), efficiently repaired these same substrates,6 phoblastoid cell line extracts. Thus, WRN may play no role in mis demonstrating that SV4O transformation alone does not inhibit mis match repair or that its functional may be redundant in match repair activity in fibrobbastoid cells. (The extract of GM637 B-bymphobbastoid cells. cells was prepared using a protocol that was slightly modified from In contrast to the results obtained with extracts of the lymphoid cell the standard methods of extract preparation described in Ref. 31.6) lines, extracts of all three WRN-mutant fibroblastoid cell lines were These results do not rule out the possibility that SV4O transformation deficient in repair of a G-G mismatch (Fig. 3). None of the fibroblast in conjunction with the presence of WRN mutations confers a mis extracts suppressed the mismatch repair activity of repair-proficient match repair deficit in fibroblasts. extracts, indicating that the repair deficiency of the fibroblast extracts Complementation of Repair-deficient Extracts. In an attempt to is not due to a trans-acting inhibitor of repair. The observations that determine the defects responsible for the mismatch repair deficiencies PSV8 I 1 is complemented by WS780 but not by W-V and that observed in extracts of the WS fibrobbast cell lines, we performed G-G complementation of WS780 by W-V is minimal (Table 2; 14% repair, mismatch repair reactions using pairwise combinations of mismatch a borderline value) suggest that these three fibroblast lines harbor repair-defective extracts. First, extracts of PSV81 1, W-V, and WS780 different mismatch repair defects. This possibility is supported by the dissimilar abilities of these fibroblast lines to complement extracts of 6 A. C. Clark, A. Umar, and TA. Kunkel, unpublished results. tumor cell lines with mutations in known mismatch repair genes 2958

Fig. 2. Mismatch repair in extracts of WS lymphoid cell lines. Reactions were performed and analyzed as described in â€œMate rials and Methods.â€•The efficiency of repair is calculated from the reduction in mixed bursts relative to an unrepaired mock reaction (containing distilled water rather than extract) and cx pressed as a percentage. Cell lines derived from WS-afflicted patients are italicized and unaffected pedigree controls are not (see Table 1). A, repair efficiency of G-G-A and G-G-B mis matches. The substrate containing the G-G mismatch at position 89 is designated â€œAâ€•whenthe nick is 3' to the mismatch at the 0. AvaIl site (position â€”264)or â€œBâ€•whenthe nick is 5' to the mismatch at the Bsu36 I site (position +276). (Position I is the a) 0 first transcribed base of the lacZ a-complementation sequence.) In both cases, the nick is situated in the (â€”)strand. Inset, ratio of blue to white plaques. B, repair efficiency for substrate containing 0) two extra bases (112)in the plus strand, designated (+) or in the â€˜C (â€”)strand, designated (â€”).â€œAâ€•andâ€œBâ€•referto the orientation of the nick, as described above. Repair of fl2 + A results in a blue plaque phenotype. whereas repair of fl2 â€”Byields a white plaque phenotype. Hence, repair changes the blue:white ratio in opposite directions, as shown in the inset.

HeLa SY75 SV76 ZM3O ZM33 AUS25 AUS1O OW

(Table 2). The data are consistent with the possibility that W-V may cells harboring mutations in hMSH2, /ZMSH3, hMSH6, hMLHJ, or be hPMS2-deficient and WS780 may be deficient in hPMS2, hMLH 1, hPMS2. One possible explanation for these differences is that PSV8 I I or hMSH3. Further work is required to resolve these possibilities. cells may harbor a mutation in an unknown mismatch repair gene. The most intriguing results in the present study were obtained with This is consistent with the observation that an extract of PSV8I 1 cells extracts from PSV81 1 cells. The PSV81 1 cell line exhibits chromo is not demonstrably deficient in any of five known mismatch repair somal instability and has an elevated HPRT mutation rate and a proteins (Table 2). Alternatively, it is possible that PSV8I I cells deletion mutator phenotype, properties that may reflect the deficiency retain a bevel of mismatch repair activity that is too low to be scored in mismatch repair activity in extracts observed here (Fig. 3). How above the noise of the extract activity assay used here (â€”10%, see ref. ever, PSV81 1 does not display detectable microsatellite instability at 31). any of five bocithat have thus far been examined (37). Cumulatively, It is remarkable that three of three WS fibroblastoid cell lines the properties of PSV81 1 cells are distinct from those observed in examined were repair deficient, whereas four of four WS bymphoid

I flfl . --@@-- 5 0 U G/G-A @ G/G-B 80

Fig. 3. Mismatch repair in extracts of WS fibroblast cell lines. Repair efficiency of G-G-A and G-G-B mismatches. Reactions were performedandanalyzedasdescribedinthe legendto Fig. 1.Inset, Lrn.ur:::::iI..@[email protected][email protected] blue:white ratios of the G-G reactions. All extracts were competent 0. HeLa pSV811 WV-i WS780 Mock for SV4O origin-dependent replication. with incorporation values exceeding by at least 10-fold those obtained in control reactions containing extract but no T antigen. The HeLa cell extract (50 @sg) incorporated 278 pmol of total nucleotide in a 2-h reaction; the comparative replicative capacities of WS cell extracts, expressed as a percentage of the HeLa extract, were: W-V, 15%; WS780, 13%; and PSV81I, 75%. 20

0 HeLa pSV8ii WV-i WS780

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. MISMATCH REPAIR IN WERNER SYNDROME CELLS cell lines were repair proficient. These results are consistent with at 15. Fukuchi, K-I., Martin, G. M., and Monnat, R. J., Jr. Mutator phenotype of Werner least two possibilities. One is that the WRN gene does participate in syndromeischaracterizedbyextensivedeletions.Proc.Natl.Acad.Sci. USA,86: 5893â€”5897,1989. mismatch repair, but its function is redundant in B-lymphobbastoid but 16. Fukuchi, K-I., Tanaka, K., Kumahara, Y., Manimno, K., Pride, M. B., Martin, G. M., not fibroblastoid cells. Another is that a mutation in the WRN gene and Monnat, R. J., Jr. Increased frequency of 6-thioguanine-resistant peripheral blood may predispose some, but not all, cell types to secondary mutations in lymphocytes in Werner syndrome patients. Hum. Genet., 84: 249â€”252, 1990. 17. Monnat, R. J., Jr. Werner syndrome: molecular genetics and mechanistic hypotheses. mismatch repair genes that bead to loss of mismatch repair activity. An Exp. Gerontol., 27: 447â€”453,1992. elevated mutation rate in WS cells, as exemplified by the elevated 18. Martin, G. M., Sprague, C. A., and Epstein, C. J. Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. Lab. Invest., 23: 86â€”92, HPRT mutation rate and the deletion mutator phenotype of the 1970. PSV81 1 cell line (15), could give rise to such secondary mutations. 19. Poot, M., Hoehn, H., Ruenger, T. M., and Martin, G. M. Impaired S-phase transit of This possibility is intriguing given the strong link between loss of Werner syndrome cells expressed in lymphoblastoid cell lines. Exp. Cell Res., 202: 267â€”273,1992. mismatch repair activity and tissue-specific tumorigenesis and the 20. Friedberg, E. C., Walker, G. C., and Siede, W. Human hereditary diseases with restricted spectrum of neoplasms observed in WS patients (2). It defective processing of DNA damage. In: DNA Repair and Mutagenesis, pp. 633â€” should be possible to test whether a cell lineage-specific expression of 671. Washington, DC: ASM Press, 1995. 21. Eshleman, J. R., and Markowitz, S. D. Microsatellite instability in inherited and functional deficits occurring in conjunction with mutations at the sporadic neoplasms. Curr. Opin. Oncol., 7: 83â€”89,1995. WRN locus contributes to the unusual tumor spectrum and the differ 22. Kolodner, R. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev., ent types of cutaneous and soft tissue pathology displayed by WS 10: 1433â€”1442,1996. 23. Umar, A., and Kunkel, T. A. DNA replication fidelity, mismatch repair and genome patients. instability in cancer cells. Eur. J. Biochem. 238: 297â€”307,1996. 24. Modrich, P., and Lahue, R. 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Samuel E. Bennett, Asad Umar, Junko Oshima, et al.

Cancer Res 1997;57:2956-2960.

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