HNPCC-like cancer predisposition in mice through simultaneous loss ...

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HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions


Niels de Wind1,2, Marleen Dekker1, Nanna Claij1, Léon Jansen1, Yvonne van Klink1, Miroslav Radman3, Greg Riggins4, Martin van der Valk1, Karin van ‘t Wout1 & Hein te Riele1

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair1 (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSα (refs 2–5), a dimer of MutS homologues MSH2 and MSH6; and MutSβ (refs 2,6,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity8–10. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice11–13 and men14–16, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families17,18. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6-deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3.

a

Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

We inactivated mouse Msh3 and Msh6 in embryonic stem (ES) cells by insertion of hygromycin and puromycin resistance markers, respectively (Fig. 1a,b). Msh3+/tm1Htr (Msh3+/hyg) and Msh6+/tm1Htr (Msh6+/pur) ES cells were then grown at high concentrations of hygromycin B or puromycin, respectively, to select for cell clones carrying two copies of the targeted allele with concomitant loss of the wild-type allele. Subsequently, Msh6 was disrupted in Msh3tm1htr/tm1htr ES cells. Msh3 and Msh6 proteins were absent in Msh3tm1htr/tm1htr and Msh6tm1htr/tm1htr ES cells, respectively (Fig. 1c). Both proteins were absent in Msh3–/–Msh6–/– cells. Thus, the targeted alleles will hereafter be indicated as Msh3– and Msh6–. Msh3 and Msh6 proteins were also nearly absent in Msh2–/– ES cells11, indicating that the stability of both proteins is dependent on their interaction with Msh2. Similar observations were made in human cells6,7. Msh2 protein level was reduced in Msh6–/– cells and further reduced in Msh3–/–Msh6–/– cells, indicating that the stability

c Msh3

b Msh6

Fig. 1 Genetic inactivation of the mouse genes Msh3 and Msh6. a, Targeting vector to insert a hygromycin resistance marker into exon 2 of Msh3. The position of exons was derived from ref. 28. b, Targeting vector to exchange part of exon 4 of Msh6 for a puromycin resistance marker. The positions of external probes and restriction sites to monitor correct targeting events are indicated. RV, EcoRV. c, Western blots prepared from wild-type, Msh2–/–, Msh3–/–, Msh6–/– and Msh3–/–Msh6–/– ES cells were probed with antibodies against full-length Msh2 or Msh6, or the carboxy terminus of Msh3. Antibodies against amino-terminal Msh3 gave similar results (data not shown).

1Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands. 2Present address: Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlands. 3Faculté de Médecine Necker (INSERM E9916), Université Paris V, Paris, France. 4Department of Pathology, Duke University Medical Center, Durham, North Carolina USA. Correspondence should be addressed to H.t.R. (e-mail: [email protected]).

nature genetics • volume 23 • november 1999

359

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a

of Msh2 depends on its interaction with Msh3 and Msh6. This shows that MutSα complexes are present at wild-type levels in Msh3–/– cells, and MutSβ complexes are present at wild-type levels in Msh6–/– cells. MutSα is in large excess to MutSβ or free Msh2 in wild-type cells. We confirmed functional inactivation of Msh3 and Msh6 by monitoring binding activity in cell extracts to oligonucleotides carrying a G•T mismatch or an unpaired TG dinucleotide. Such activity was detected in wild-type ES cells and was dependent on Msh2 but not Msh3, suggesting that Msh2 and Msh6 are the major constituents (Fig. 2a). Residual binding activity to both types of mismatches was present in Msh6–/– cells, but disappeared on concomitant inactivation of Msh3. Thus, both MutSα and MutSβ can bind to G•T and extrahelical dinucleotide mismatches, whereas free Msh2 can not. Although MutSα activity exceeds that of MutSβ, on an equimolar basis the two complexes appear to have similar mismatch-binding affinities. We next introduced the Msh3– and Msh6– alleles into the germ line of mice. We then compared survival and tumour incidence in Msh3–/–, Msh6–/– and Msh3–/–Msh6–/– mice with those previously found in Msh2–/– mice13. Msh6–/– mice were highly cancer prone, approximately 75% succumbed within one year (Fig. 3) and virtually all presented with lymphoma or epithelial cancer of the uterus and skin (Table 1). Most lymphomas appeared within 30 weeks; in this period the incidence in Msh6–/– animals (9/12) was as high as that in Msh2–/– animals (31/39; ref. 13). In contrast to Msh2–/– animals13, Msh6–/– mice rarely developed intestinal tumours: 2 of 22 animals at a mean age of 52 weeks (Table 1).

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Fig. 2 Mismatch-binding activity in Msh mutant ES cells. a, A gel retardation assay was used to measure binding activity in extracts from wild-type, Msh2–/–, Msh3–/–, Msh6–/– and Msh3–/–Msh6–/– ES cells to radioactively labelled doublestranded oligonucleotides containing no mismatch (Ho), a G•T mismatch (G•T) or an unpaired dinucleotide (+2). b, Binding activity in each extract to an oligonucleotide containing an E2F site was used as a quality and loading control. Arrows indicate the positions of protein/DNA complexes.

Msh3 deficiency did not predispose to cancer, nor did it accelerate lymphomagenesis in Msh6–/– animals (Fig. 3 and Table 1). Combined Msh3/Msh6 deficiency, however, predisposed to development of intestinal tumours which were found in 8 of 13 animals at a mean age of 30 weeks (Table 1). In addition to mutation avoidance, we have previously demonstrated two other functions of MMR in ES cells11: Msh2 mediates toxicity of methylating agents and is required to suppress homologous recombination between slightly diverged (