Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

Agnes Baross-Francis, M.Kate Milhausen, Susan E. Andrew2, Gareth Jevon1 and Frank R. Jirik3

Centre for Molecular Medicine and Therapeutics, Department of Medicine and
1 Department of Pathology, University of British Columbia, Vancouver, BC V5Z 4H4 and
2 Department of Medical Genetics, University of Alberta, AB T6G 2H7, Canada


    Abstract
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 Abstract
 Introduction
 References
 
We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2-/-, exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2-/- tissues. To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2. While lacI gene hypermutation was again clearly evident in Msh2+/-Pms2-/- and Msh2-/-Pms2-/- thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice. The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.

Abbreviations: MMR, mismatch repair.


    Introduction
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 Abstract
 Introduction
 References
 
As tumor initiation and progression requires a variety of specific genetic changes, at a pace exceeding that provided by the spontaneous mutation rate, it has been hypothesized that mutator phenotypes must be a common feature of neoplasia (1). This hypothesis has now been supported by the finding of increased rates of genomic instability (e.g. instability at repetitive sequences or chromosomal aberrations) within a great variety of tumor types (2). The high frequency of silent, unselected mutations in genes such as p53 also provides evidence of an increased mutation rate of cancer cells (3). Genomic instability which is commonly exhibited by tumors can be linked to changes in processes that regulate the fidelity of DNA polymerases or the effectiveness of DNA repair pathways (4). Mutations in one of four human DNA mismatch repair (MMR) genes (the MutS ortholog hMSH2 and the MutL orthologs hMLH1, hPMS2 and hPMS1) are commonly found in the autosomal dominant cancer syndrome, hereditary non-polyposis colorectal cancer (HNPCC). This syndrome is characterized not only by early onset colon carcinomas, but also by malignant tumors of the endometrium, stomach, upper urinary tract, small intestine and ovary (5). Tumors arising within individuals with this syndrome, who are, in most cases, heterozygous for a germline mutation in an MMR gene, exhibit inactivation of the second allele of the same repair gene (6). Homozygous germline inactivation of an MMR gene has recently been reported in two families. In these cases, hMLH1-deficient children developed hematological malignancies and neurofibromatosis type 1-like features at an early age (7,8). Cells deficient in specific MMR components may show not only microsatellite instability, but also exhibit dramatic increases in the frequency of base substitutions in tumor lines (5).

MMR-deficient mice generated via gene targeting provide model systems for exploring the consequences of MMR deficiency. In contrast to human HNPCC, mice lacking one allele of the Msh2 gene tend not to experience an increased rate of tumor development within their lifetime. Homozygous Msh2-/- mice, on the other hand, exhibit an increased incidence of various types of tumors, including lymphomas (predominantly of the thymus), small intestinal adenomas and carcinomas, and squamous cell tumors (6,9,10). Such model systems clearly demonstrate that MMR deficiency is associated with carcinogenesis; however, it is interesting to note that tumors arise only within a restricted subset of tissues, and with varying latencies, despite a global lack of MMR activity (6,9,10).

DNA obtained from the tissues of mice lacking Msh2 and transgenic for the lacI mutational reporter gene, demonstrate an increase (~10-fold) in spontaneous mutation frequency as compared with Msh2 heterozygotes, or wild-type mice (11). Interestingly, Msh2-/- murine thymic lymphomas revealed greatly elevated mutational frequencies compared with normal Msh2-/- tissues, as well as mutation clusters within lacI genes recovered from these tumors (12).

Similar to Msh2 deficiency, mice having a targeted disruption of Pms2 also demonstrate an elevated rate of tumorigenesis, consisting primarily of thymic lymphomas and miscellaneous sarcomas (13). Interestingly, and unlike Msh2-deficient mice, a lack of PMS2 does not appear to lead to intestinal epithelial tumor formation (5,13). Also, unlike Msh2-deficient mice, Pms2-/- males are sterile due to a defect in chromosome pairing during meiosis (14). Such findings demonstrate that DNA repair genes lying in common pathways could play roles in processes that are unrelated to MMR. Similar to Msh2-/-, Pms2-/- mice demonstrate elevated mutation frequencies in all tissues examined, as compared with Pms2+/-, or wild-type mice, as determined using either the supF (15), or lacI (16) transgenic reporter systems.

To establish whether the lacI gene hypermutation previously observed in Msh2-/-lymphomas (12) would also be seen in lymphomas of other MMR-deficient mice, lymphomas of Msh2+/-Pms2-/– and Msh2-/-Pms2-/- double mutant mice were examined using the lacI system. The lacI gene mutation frequencies obtained were similar to those reported previously for Msh2-/- lymphomas (12). In contrast, three of four advanced non-thymic tumors arising within Msh2-/- and Pms2-/- mice failed to show lacI gene mutation frequency elevations, suggesting that hypermutation is not a consistent feature of tumors arising in MMR-deficient mice.

Msh2+/– (9) and Pms2+/– (14) mice were crossed with the BC-1 lacI transgenic line. Hemizygous BC-1 mice carry approximately 30 copies of a {lambda}-phage shuttle vector transgene containing the lacI reporter gene (17). Msh2 or Pms2 heterozygous lacI+mice were bred to obtain lacI+ knockouts. In order to produce Msh2 and Pms2 double knockout lacI+ mice, Msh2 and Pms2 knockout or heterozygous lacI+ parents were used, except for Pms2–/– males, which are sterile (14). For genotyping, 0.5 cm tail clips, obtained from anesthetized mice, were digested in 300 µl lysis buffer containing 1.2 mg/ml proteinase K, 50 mM Tris–HCl pH 8.0, 10 mM EDTA, 0.1% SDS and 100 mM NaCl. Following heat- inactivation of the proteinase, 1 µl of a 20-fold dilution was used in PCR reactions to determine the Msh2 (9), Pms2 (14) and lacI genotypes (17). MMR-deficient mice that appeared moribund were killed by CO2 inhalation and tumor type was established by necropsy and histological examination of hematoxylin and eosin-stained tissue sections. Mice were viral antibody free and housed in a barrier facility according to institutional guidelines.

Isolation of tissues and transgenic {lambda}-phage rescue were carried out as described (18,19). Phage genomes were excised from chromosomal DNA and packaged using Transpack (Stratagene) phage packaging extract. Rescued phage were then plated on SCS-8 (Stratagene) bacterial lawns containing X-gal, and lacI mutation frequencies obtained by determining the ratio of mutant (blue) to non-mutant (colorless) plaques. Mutation frequencies were determined from 4–10 packaging reactions per sample. The mutation frequency variation between different packaging reactions was not bigger than between plates from the same packaging reaction (0.5–2-fold differences from final result). Phage containing lacI mutations were confirmed as described previously (17). Following isolation of mutant clones, lacI genes were amplified by PCR and purified using QIAquick DNA purification kit (Qiagen). Templates obtained from randomly selected lacI mutants were sequenced using primers that spanned the lacI gene (17) with an ABI 388 DNA sequencing instrument (Applied Biosystems).

Mutation frequencies obtained from thymic lymphomas of two Pms2-/-Msh2-/-/lacI mice, and one Pms2-/-Msh2-/+/lacI mouse (Table IGo; Figure 1Go) were within the same range as the lacI mutation frequencies we reported previously for six Msh2-/- thymic lymphomas (12). Thus, the thymic lymphoma mutation frequencies of mouse S (Pms2-/-Msh2-/+/lacI), Q and R (Pms2-/-Msh2-/-/lacI) were elevated 6.0-, 4.1- and 17.3-fold, respectively, as compared with brain tissue from the corresponding host (Tables I and IIGoGo). As lymphoma cell infiltrates in the mouse brain are either minimal or absent by histological examination (12), this tissue was selected as a control for `background' lacI mutation frequencies unique to each animal. Brain mutation frequencies are similar to those of other tissues in Msh2-/- mice, including thymus (11,12). To estimate the potential effect of clonality on the mutation frequencies of these thymic lymphomas, ~10 (911) randomly selected mutants were characterized for each tumor (data not shown). Tables I and IIIGoGo show that after correction for clonality, mutation frequencies remained elevated. These findings clearly mirror the results obtained in our previous study of Msh2-/- thymic lymphomas, where lacI gene mutation frequency elevations in the 3.2- to 17.4-fold range were seen (12). Including the three thymic lymphomas in the current study, all nine consecutive thymic lymphomas analyzed to date show elevated lacI gene mutation frequencies (`hypermutation').


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Table I. Spontaneous lacI mutation frequencies: Msh2-/-Pms2-/- and Msh2+/-Pms2-/- thymic lymphomas
 


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Fig. 1. lacI mutation frequencies of DNA MMR-deficient tumors (gray bars are based on plating data and black bars show mutation frequencies after correction for clonality), and for corresponding brains (white bars). Each cluster of three bars represents one mouse. (The tumor types are: DL, diffuse lymphoma; SCC, squamous cell carcinoma; OS, osteogenic sarcoma; TL, thymic lymphoma.)

 

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Table II. Spontaneous lacI mutation frequencies: Msh2-/-Pms2-/- and Msh2+/-Pms2-/- brains
 

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Table III. Spontaneous lacI mutation frequencies: non-thymic tumors deficient for MSH2 or PMS2
 
Although the numbers of mice belonging to each genotype in this study were relatively small, the results show that deficiencies of Msh2, Pms2 or both of these MMR genes, lead to similar lacI mutation frequencies in thymic tumors and in normal tissues. In keeping with current models of MSH2 and PMS2 functioning within the DNA MMR pathway (20), brains of the double mutant (Pms2-/- and Msh2-/-) mice Q and R, revealed lacI mutation frequencies that were within the range of those encountered in single mutant mice (11,12,16).

We next sought to establish whether the lacI gene `hypermutation' observed in the MMR-deficient thymic lymphomas would prove to be a common feature of all tumors in these mice. To evaluate this possibility, lacI mutation frequencies were determined for four non-thymic tumors obtained from Msh2-/- mice (Table IIIGo; Figure 1Go). Interestingly, three out of four non-thymic tumors (L, an ~2 cm3 diffuse extra-thymic lymphoblastic lymphoma, probably of B-cell origin, found on the shoulder; M, an ~1.5 cm3 squamous cell carcinoma arising from the skin at the groin; N, an ~1.5 cm3 osteogenic sarcoma arising from the lower femur on one of the hind legs), failed to show lacI mutation frequency elevations when compared with the corresponding brains of each affected animal (Tables III and IVGoGo; Figure 1Go). Indeed, the frequencies of these three tumors were within the same range as normal Msh2-/- or Pms2-/- tissues (11,12,16). Tumor P (a diffuse extra-thymic lymphoma, probably of B-cell origin, found on the wall of small intestine), in contrast, exhibited an ~45-fold lacI mutation frequency increase compared with brain from the same host (Tables III and IVGoGo; Figure 1Go), suggesting that peripheral lymphomas may be heterogeneous with respect to the `hypermutator' phenotype. After correction for clonality, the increase seen in tumor P was still ~13-fold, a value comparable with that of the thymic lymphomas (12). Thus, elevated lacI mutation frequencies are not a feature of every Msh2-/- tumor, but instead appear to depend on tumor type, or possibly subtype (in the case of the peripheral lymphomas).


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Table IV. Spontaneous lacI mutation frequencies: brains deficient for MSH2 or PMS2
 
Spontaneous lacI mutation frequency elevations of 5- to 15-fold are typical of Msh2-/-/lacI mouse tissues as compared with control mice (11), and similar differences are seen when Pms2-/- and control mice are compared (16). Interestingly, both Msh2-/- and Pms2-/- thymic lymphomas demonstrated additional striking increases in lacI mutations, perhaps consistent with some type of `hypermutator' mechanism (12). In contrast, the non-thymic tumors analyzed, despite being of a size and degree of progression (metastatic deposits in multiple organs) comparable with the lymphomas (data not shown), revealed lacI mutation frequencies that were within the range of those observed in normal DNA MMR-deficient tissues (11,12). What factor(s) is responsible for the dramatic increases in mutation frequency seen in the MMR-deficient lymphomas? One possibility is that the lacI mutation rate within the thymic tumors is similar to that of normal tissues, and that the lacI genes simply accumulate mutations as a function of the number of cell divisions within the tumor. As there is no method for accurately quantifying the number of cell divisions required to generate a normal tissue or a tumor, in vivo mutation rates cannot be determined with any level of confidence. However, higher numbers of cell divisions do not necessarily lead to increased lacI mutations, as an increased lacI mutation frequency was not observed in all the DNA MMR-deficient tumors we analyzed. It is still plausible, however, that the MMR-deficient thymic lymphomas undergo much greater levels of proliferation and apoptosis during their growth than the non-thymic tumors (L, M, N), thus accounting for their elevated mutation frequencies. On the other hand, when lacI mutation frequencies of p53-/- thymic lymphomas were examined, only one out of eight thymic tumors showed an increase over the control tissue (21,22). Mitotic counts performed in four of these thymic lymphomas, including one which showed an increased mutation frequency, revealed no apparent correlation between the mitotic index and lacI mutation frequency (22). This finding supports our contention that cell proliferation may not be the only mechanism responsible for the increased mutation frequencies observed in the nine thymic lymphomas we have analyzed thus far.

Multiple mutations in single lacI genes appeared in the thymic lymphomas of mice Q, R and S with a higher frequency than in normal tissues. Out of 32 mutants sequenced from these tumors, 28 mutations were independent (data not shown), three of which contained mutational clusters (Table VGo). So far only one such cluster was found among lacI mutants from 67 Pms2-/- and 39 Msh2-/- normal thymuses, (12,16); thus, the frequency of multiple mutations is significantly higher in the thymic lymphomas ({chi}2 test, P < 0.05). Among the 42 independent mutants (out of 49 sequenced; data not shown) from the non-thymic tumors, there was no lacI gene with a multiple mutation. The finding of multiple, often non-randomly clustered lacI mutations in a number of the phage rescued from the previously examined thymic lymphomas of MMR-deficient mice (12), and in the current study (Table VGo, tumors Q, R and S), suggests a mutational mechanism beyond that simply afforded by increased numbers of cell divisions. Such a mutator could arise in various ways, such as: alterations in nucleotide pools due to mutations in synthetic pathways or specific nutrient deficiencies, DNA polymerase gene mutations, or defects in other DNA damage recognition and repair components (4). Whether the hypermutation seen in the thymic tumors provides any advantage with respect to tumorigenesis and progression, or whether it merely represents a gratuitous mutational process non-essential for either of these processes remains to be established. It is tempting to speculate, however, that activation of such a mutator mechanism (perhaps stemming from mutation of a specific gene caused by the absence of DNA MMR), as an early event within a developing thymocyte might promote the increased rate of tumorigenesis observed in this tissue.


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Table V. Multiple mutations occurring within single lacI genes rescued from Msh2-/-Pms2-/- (Q and R), and Msh2+/-Pms2-/- (S) thymic lymphomas
 


    Notes
 
3 To whom correspondence should be addressed Email: jirik{at}cmmt.ubc.ca Back


    Acknowledgments
 
We are very grateful to R.M.Liskay for providing the Pms2-/- mice, and to T.W.Mak and A.Reitmair for the Msh2-/- mice. We thank J.E.Penney and L.A.Waddleton for maintaining the various mouse lines, and N.Makhani and K.Fichter of the Canadian Genetic Disease Network DNA Sequencing Core Facility in Vancouver. This study was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society (to F.R.J.). S.E.A. was the recipient of a Medical Research Council of Canada Post-Doctoral Fellowship award, and A.B.-F. held a Natural Sciences and Engineering Research Council of Canada Graduate Scholarship.


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Received October 29, 1999; revised February 8, 2000; accepted February 17, 2000.