Loss of Heterozygosity and Point Mutation at Aprt Locus in T Cells and ®Broblasts of Pms27/7 Mice

Loss of heterozygosity and point mutation at Aprt locus in T cells and ®broblasts of Pms27/7 mice

Changshun Shao1, Moying Yin2, Li Deng1, Peter J Stambrook3, Thomas Doetschman2 and Jay A Tisch®eld*,1

1Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, NJ 08854-8082, USA; 2Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267-0521, USA; 3Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267-0521, USA

Mice null for the Pms2 mismatch repair (MMR) gene tion (Harfe and Jinks-Robertson, 2000). Third, MMR exhibit a predisposition to lymphoma, microsatellite plays a role in the regulation of apoptosis (Gong et al., repeat instability, and failure of spermatogenesis. To 1999; Toft et al., 1999; Zhang et al., 1999; Zeng et al., study the role of Pms2 in the maintenance of in vivo 2000). In the absence of MMR, apoptosis is genomic integrity in somatic cells, we characterized Aprt compromised in cells with DNA damage, and the mutations in T cells and ®broblasts of 1296C3H increased chance of survival and proliferation of those Pms27/7Aprt+/7 mice. The spontaneous frequency of cells may allow further accumulation of mutations. DAP-resistant T lymphocytes, as a consequence of Organisms and cells that are MMR-de®cient typi- APRT-de®ciency, was increased threefold. Point muta- cally exhibit an increased level of genetic instability, or tion, which accounted for less than 20% of the DAPr a mutator phenotype. The mutator phenotype includes mutant clones in Pms2+/+ mice, was predominant in the instability of microsatellite repeats, elevation of point mutant T cell clones from Pms27/7 mice. These point mutation and increase of recombination between mutations were predominantly TA to CG transitions. divergent sequences (homeologous recombination). In Fibroblasts of Pms27/7 mice exhibited only a modest humans, MMR-de®ciency can lead to several types of increase in the frequency of clones with point mutations, cancer. For example, inherited mutations in a subset of such that mitotic recombination was still the primary MMR genes, including MSH2, MLH1 and PMS2, are cause of APRT de®ciency. Thus, the mutator phenotype responsible for hereditary non-polyposis colorectal as a consequence of PMS2 de®ciency is tissue-dependent, cancer (HNPCC). which may be related to the tissue-speci®c tumor To elucidate the biological functions of the MMR proneness of Pms27/7 mice. genes in vivo, mouse mutants of various MMR homologs Oncogene (2002) 21, 2840 ± 2845. DOI: 10.1038/sj/ have been generated through targeted gene disruption in onc/1205358 embryonic stem cells. Depending on the MMR gene, the homozygous mutant mice displayed one, two or all of Keywords: mismatch repair; PMS2; APRT; mouse the following three phenotypes (Buermeyer et al., 1999): model; somatic mutation; loss of heterozygosity (i) tumor predisposition, (ii) mutator phenotype, and (iii) meiotic defect(s). For example, Mlh17/7 mice exhibit all three phenotypes, whereas Pms17/7 mice only show a Introduction mild mutator phenotype. Most studies of the mutator phenotype in MMR- DNA mismatch repair (MMR) proteins contribute to de®cient mice were based on observations of micro- the maintenance of genomic integrity in multiple satellite repeat markers (Yao et al., 1999) or bacterial pathways (Modrich and Lahue, 1996, Kolodner and transgenes, such as supF (Narayanan et al., 1997), lacI Marsischky, 1999; Buermeyer et al., 1999, Harfe and and cII (Andrew et al., 1997, 2000; Baross-Francis et Jinks-Robertson, 2000). First, MMR corrects mis- al., 2001). However, microsatellite repeat markers only paired or extrahelical nucleotides that are infrequently re¯ect frameshift changes in sequences containing introduced during DNA replication, by mis-incorpora- mono- or dinucleotide repeats. While bacterial trans- tion or by slippage. Second, MMR-mediated suppres- genes can show base substitutions, frameshifts and sion of recombination is believed to reduce the other intragenic alterations, there are concerns as to incidence of both translocation and mitotic recombina- whether or not their behavior mimics that of endogenous genes since they usually reside as multiple copies in a host cell, are hypermethylated and transcriptionally inactive (Mirsalis et al., 1995). We *Correspondence: JA Tisch®eld; E-mail: [email protected] Received 3 December 2001; revised 21 January 2002; accepted 22 have documented the use of an endogenous, and February 2002 ubiquitously expressed, APRT (adenine phosphoribo- Somatic mutations in Pms27/7 mice C Shao et al 2841 syltransferase) gene as a reporter for loss of hetero- The shift of the mutational spectrum between the two zygosity (LOH) in vivo (Shao et al., 1999, 2000, 2001). groups is highly signi®cant (P50.0001). Analysis of Because Aprt is autosomal, it can detect most genetic ¯anking SSLP showed that almost all of the class I alterations at the chromosome level, such as mitotic variants in either Pms2+/+ or Pms27/7 mice were recombination and multi-loci deletion, in addition to derived from mitotic recombination (Table 2). The intragenic mutations and small deletions. In this study, frequency of such clones, as calculated by multiplying we characterized somatic mutations at Aprt in the median frequency of DAPr clones with the fraction normal T lymphocytes and ®broblasts of 1296C3H of mitotic recombination in each group, was Pms27/7Aprt+/7 mice. 19.261076 and 25.761076 in Pms2+/+ and Pms27/7 mice, respectively. This indicates that mitotic recombination in T cells are not aected by the loss of PMS2. The median frequency of class II T cells, on the Results other hand, diers dramatically between Pms2+/+ and Pms27/7 mice, 2.961076 vs 47.661076, a 16-fold T cells of Pms27/7 mice exhibit increased point mutation dierence. Class II variants can either be caused by at the Aprt and Hprt loci point mutation or by epigenetic silencing of the wild- Though the frequency of DAP-resistant T cells varied type (untargeted) Aprt allele (Shao et al., 1999, Rose et greatly within each group, Pms27/7 mice exhibited a al., 2000). We sequenced all ®ve exons, part of the higher frequency than Pms2+/+ mice (P=0.017, promoter region and three introns of the Aprt gene in Mann ± Whitney U-test). The median frequency was 63 clones recovered from Pms27/7 mice and detected increased threefold in Pms27/7 mice, from 22.661076 point mutations in 51. This indicates that the increase to 73.361076 (Figure 1). in the frequency of DAPr T cells was primarily the We divided the DAPr clones into two classes consequence of point mutation. Strikingly, more than according to the absence or retention of the untargeted half of the mutations in Pms27/7 mice were T to C Aprt allele (Shao et al., 1999). Class I variants, those transitions (Table 3). This predominance of TA to CG exhibiting loss of the untargeted Aprt, were predomi- transitions is consistent with the mutational spectrum nant in Pms2+/+ mice (40/46, 87%), which is of Hprt in Pms27/7 mice (Shaddock et al., 2001), but consistent with our previous study (Shao et al., 2000). is in contrast to the mutational spectrum observed in However, class I clones, those retaining the untargeted lacIofPms27/7 mice (Andrew et al., 1997, Baross- allele, replaced class I as the primary cause of APRT- Francis et al., 2001), in which CG to TA transitions de®ciency in Pms27/7 mice (110/170, 65%) (Table 1). predominate. In addition to base substitution, eight frameshift mutations at di- or mononucleotide runs were detected in class II clones recovered from Pms7/7 mice (Table 3, Figure 2). Four of the 51 (8%) clones that were con®rmed to have point mutation were putative sib clones (having the same mutation), indicating that most of mutants had originated independently. Thus, the higher frequency of T cell variants in Pms27/7 mice is caused by an increased mutation rate. We also estimated the frequency of 6-TG resistant (HPRT-de®cient) T cells in Pms27/7 and Pms2+/+ mice. Since Hprt is X-linked, HPRT de®ciency is presumably caused only by intragenic mutations. Consistent with the observations with Aprt, Pms27/7 mice also exhibited a dramatic increase in the frequency of the 6-TGr T cells, 2.3+ 1.461076 (s.e.m., n=9) in Pms2+/+ mice vs 109.8+16.361076 (n=12) in Pms27/7 mice. In comparison to the 16-fold increase in the median frequency of class II DAPr variants, which are primarily caused by point mutation, in Pms27/7 mice, the magnitude of increase for 6- TGr variants is much greater (48-fold). The large size

Table 1 Class II variants DAPr T cells in Pms27/7 mice No. mice Class I Class II Total % of class II Figure 1 Frequency of DAPr T cells in individual mouse +/+ spleens. Each spot represents one spleen. Bars represent median Pms2 9 40 6 46 13 7/7 values Pms2 15 60 110 170 65

Oncogene Somatic mutations in Pms27/7 mice C Shao et al 2842 Table 2 Mutational spectrum of class I variant clones ®broblasts in Pms27/7 mice (35/54), though the Mitotic Deletion or Total no. fraction of class I was lower in Pms27/7 mice (65%), Cell type Genotype recombination gene conversion of clones than in Pms2+/+ mice (83%) (Table 4). T cells Pms2+/+ 35 1 36 As with the T cells, most of the class I ®broblast Pms27/7 43 0 43 clones were caused by mitotic recombination in Fibroblasts Pms2+/+ 14 1 15 ®broblasts. We calculated that the frequency of mitotic Pms27/7 25 2 27 recombination is similar between the two groups of mice, 62.361076 in Pms2+/+ vs 72.261076 in Pms27/7 mice. Table 3 Point mutations in Aprt of Pms27/7 mice are primarily We also estimated the frequency of 6-TGr ®broblasts T4C transitions in Pms2+/+ and Pms27/7 mice (Table 5). The 6-TGr Pms2+/+ Pms2+/7 Pms27/7 clones were recovered from about 50% of the ears of 7/7 Transitions the Pms2 mice, in comparison to 16% in wild-type 7/7 T4C1126mice. Excluding one Pms2 outlier, the accumulative A4G3frequency of 6-TGr is about four times higher in C4T111Pms27/7 mice than in wild-type mice, 4.361076 in G4A6Pms2+/+ vs 17 x 1076 in Pms27/7. Transversions 2 3 2 bp deletions 1 1 These ®ndings with ®broblasts indicate that although 1 bp deletions 4 point mutations are more common in Pms27/7 than in 1 bp deletions 3 Pms2+/+ mice, the magnitude of increase is smaller than that in T lymphocytes. Total 5 2 47

Discussion

We estimated the frequency of in vivo DAPr and 6-TGr mutants in normal T cells and ®broblasts of Pms27/7 mice and characterized the mutational spectrum of

Figure 2 Distribution of frameshift mutations in ®ve exons of Aprt in Pms27/7 mice. Upward arrow head, deletion; downward arrow head, insertion

of the Hprt gene, 33 kb (Melton et al., 1984) vs 3kb for Aprt (Dush et al., 1985), and certain unique structures, such as the six G repeat, may make it more unstable than Aprt in the absence of MMR.

Fibroblasts of Pms27/7 mice exhibit a modest increase in point mutations at the Aprt and Hprt loci In contrast to the signi®cant increase in the frequency of DAPr mutants in T cells of Pms27/7 mice, the frequency of DAPr ®broblasts was not signi®cantly dierent between Pms27/7 and Pms2+/+ mice (P=0.16), although the median frequency was higher in Pms27/7 mice than in Pms2+/+ mice (11161076 versus 7561076) (Figure 3). Molecular characterization showed that class I Figure 3 Frequency of DAPr ®broblasts in individual mouse variants still accounted for the majority of the DAPr ears. Each spot represents one ear. Bars represent median values

Oncogene Somatic mutations in Pms27/7 mice C Shao et al 2843 DAPr cell variants. We observed a dramatic elevation repair and mutation ®xation. Since mismatches in of point mutation in T cells of Pms27/7 mice, which DNA are primarily introduced during DNA replica- signi®cantly shifted the mutational spectrum. While tion, via misincorporation or slippage, the accumula- APRT-de®ciency in T cells is predominantly caused by tion of DNA replication errors in a cell may be a mitotic recombination in Pms2+/+ mice, it is largely function of the number of prior cell divisions. Possibly, due to point mutation in Pms27/7 mice. However, skin ear ®broblasts undergo fewer cell divisions than ®broblasts only exhibit a modest increase in point peripheral T cells in vivo, so that their chances of mutation, as re¯ected by the relatively unchanged accumulating replication errors are reduced in compar- mutational spectrum of DAPr ®broblasts of Pms27/7 ison. mice. In general, the absolute frequency of clones The primary function of MMR is to maintain derived from mitotic recombination is not signi®cantly genomic integrity. Thus, it is likely that tumor dierent between Pms2+/+ and Pms27/7 mice. proneness displayed by the MMR-de®cient mice is Since the assay using Aprt as a reporter is based on driven by increased genetic instability, which can the clonal proliferation of mutant cells in vitro, we were render key tumor suppressor genes more mutable. unable to extend our observations to other tissues, such However, previous studies using microsatellite repeat as intestinal epithelium. However, with the two types and transgenic reporters showed that tumor spectrum of tissue we examined, we can conclude that the and genetic instability are not necessarily correlated. Pms27/7 mutator phenotype is tissue-dependent. Our For example, although Pms27/7 mice are predomi- ®ndings are in contrast to a study using supF or nantly predisposed to lymphomas, the mutation supFG1 transgenic mice, in which spleen and skin frequencies of supF and supFG1 are equally tissues of Pms27/7 mice exhibited equal elevation in elevated in the skin and spleen tissues of those mice mutation frequency (Narayanan et al., 1997), but are (Narayanan et al., 1997). Also, whereas both Pms27/7 consistent with a study of Msh27/7lacI transgenic mice and Mlh17/7 mice exhibit microsatellite instability in in which lacI showed a greater magnitude of increase intestinal tissues, only Mlh17/7 mice are predisposed in mutation frequency in thymus than in brain to intestinal carcinoma (Prolla et al., 1998). Thus, it is compared to Msh2+/+ mice, 15.2-fold and 4.8-fold possible that the tissue-speci®c tumor proneness in respectively (Andrew et al., 1997). MMR-de®cient mice is not solely determined by an It is unclear as to why point mutation is dieren- increased level of genomic instability. Alternatively, the tially elevated in dierent tissues of Pms27/7 mice. One transgenic and microsatellite reporters may have not possible explanation may relate to the proliferation adequately re¯ected genetic changes at endogenous history of each tissue. A recent study showed that loci. quiescent mouse cells do not repair premutatgenic Our study showed that Aprt exhibits a dierent damage and that mutation frequency is not changed spectrum of point mutations in comparison to that of during quiescence (Bielas and Heddle, 2000). DNA lacI. While more than half (55%) of the point repair and increase of mutation frequency were mutations are T to C transitions at Aprt of Pms27/7 observed only when quiescent cells were induced to mice, only 14% are caused by TA to CG transitions at proliferate. Thus, proliferation is required for both lacI (Baross-Francis et al., 2001). Also, deletions and insertions at mononucleotide runs account for 15% of the point mutations at Aprt, but such frameshift mutations account for 34% at lacI (Baross-Francis et Table 4 Class I variants predominate in DAPr ®broblasts of al., 2001). Interestingly, another endogenous reporter Pms2+/+ and Pms27/7 mice gene, Hprt, displays a similar spectrum as Aprt in Class I Class II Total % of class I Pms27/7 mice (Shaddock et al., 2001). Pms2+/+* 96 22 118 81 MMR proteins in yeast and possibly mice also have Pms2+/+ 15 3 18 83 anti-recombination activity, which suppresses recombi- Pms27/7 35 19 54 65 nation between divergent DNA sequences (Harfe and Jinks-Robertson, 2000, Shao et al., 2001). Loss of *From Shao et al. (2000) MMR in yeast usually results in increased homologous

Table 5 Distribution of 6-TGr ®bbroblasts in ears of Pms2+/+ and Pms27/7 mice Pms2+/+ Pms27/7 No. No. ears No. 6-TGr No. No. ears No. 6-TGr CFU ears with 6-TGr colonies ears with 6-TGr colonies

20 000 ± 40 000 4 0 0 6 1 5 40 001 ± 60 000 6 1 4 12 8 9 60 001 ± 80 000 3 1 1 8 4 11 480 000 5 1 1 3 2 448

Total 18 3 (16.7%) 6 29 15 (52%) 473

CFU, colony-forming unit

Oncogene Somatic mutations in Pms27/7 mice C Shao et al 2844 recombination (Harfe and Jinks-Robertson, 2000). It (2 mg/ml) or 2,6-diaminopurine (DAP) (50 mg/ml). Their appears from the present study that mitotic recombina- colony-forming eciency (CFE) was estimated by seeding tion between the two homologues of chromosome 8, four cells per well in the presence of feeder cells, which are derived from strain 129 and strain C3H, respectively, is stimulated splenocytes irradiated with 6000 rads of X-rays. not aected in Pms27/7 mice. Probably, PMS2 is not Mutant T cell colonies were scored on day 10. The mutant frequency was calculated by applying Poisson statistics. involved in the recognition and processing of the Skin ®broblasts were prepared as described (Shao et al., recombination heteroduplex during mitotic recombina- 1999). They were seeded in 100 mm plates (16106 cells/plate) tion. Alternatively, the sequence dissimilarity between containing 6-TG (5 mg/ml) or DAP (50 mg/ml) for recovery of the two strains may not be sucient to produce a drug-resistant cell colonies. Their CFE was estimated by signi®cant inhibitory eect on recombination. Studies plating 5 000 to 10 000 cells in the presence of 16106 with other MMR gene knockout mice, such as irradiated ®broblasts. Mutant ®broblast colonies were scored Msh27/7 and Mlh17/7 mice, will address these ques- on day 12. tions. Molecular analysis Characterization of DAPr clones was as described (Shao et al., 1999). Brie¯y, DAPr clones were ®rst divided into two Materials and methods classes by the absence (class I) or retention (class II) of the untargeted allele. Class I clones were further characterized Mice with polymorphic microsatellite markers along chromosome Production of Pms2-de®cient and Aprt-de®cient mice (both 8 to determine their mechanism(s) of origin. Clones that using D3 embryonic stem cells of 129/Sv mouse origin) has exhibited LOH at loci distal to Aprt but remained been described (Baker et al., 1995, Engle et al., 1996). The heterozygous at loci proximal to Aprt were interpreted to targeted (mutant) Aprt allele contains a neo insertion in exon be derivatives of mitotic recombination, as was previously 3, making it easily distinguishable from the wild-type allele. demonstrated more rigorously (Shao et al., 1999). Clones that Pms2+/7 mice in N5C57 background were backcrossed to did not exhibit LOH at the most distal microsatellite marker 129/Sv and C3H/HeJ, respectively, for four generations to locus, D8Mit56, were interpreted to be the outcome of gene produce (N4)129Pms2+/7 and (N4)C3HPms2+/7 mice. conversion or interstitial deletion. Class II clones were 1296C3HAprt+/7 hybrid mice with dierent Pms2 geno- sequenced for all ®ve exons, introns 1, 3 and 4, and part of types were generated by crossing (N4)129Pms2+/7Aprt+/7 the promoter region of the untargeted Aprt allele for mice to (N4)C3HPms2+/7mice. Aprt and Pms2 genotyping detection of point mutation. were done as described (Shao et al., 1999; Baker et al., 1995). The hybrid mice were 2 to 3 months old when sacri®ced.

Isolation of mutant clones Splenocytes were isolated as described (Meng et al., 1998). Acknowledgments After being stimulated with conconavalin A overnight, the We thank RM Liskay for generously providing us with splenocytes were seeded in 96-well plates at the density of Pms2 knockout mice. This work was supported by grants 26104 cells/well in the presence of 6-thioguanine (6-TG) from NIH (R01DK38185, P01ES05652 and P30ES05022).

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