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

Abstract

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, 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 families. 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. 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.

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

“…Therefore, we have introduced the target 1 neo reporter into the Rosa26 locus of Msh3 À/À cells and compared the efficiency of the +1 and +4 oligonucleotides in these cells to their performance in wild-type and Msh2 À/À cells. Figure 2b shows that, as expected, the +1 oligonucleotide performed as poorly in Msh3 À/À cells as in wild-type cells, confirming the specificity of the MSH2/6 complex (which is still present in Msh3 À/À cells, De Wind et al 22 ) for small mismatches. In contrast, all +4 oligonucleotides, except +4-2, significantly improved in Msh3 À/À cells compared to wild-type cells (P-values for oligos +4-7, -3, -1, -5, -10, -4, -9 o0.002; for oligos +4-8, -6, -12 o0.02), in about half of the cases (oligos +4-7, -3, -1, -5, -10 and -4), reaching a frequency that was only two-to threefold lower as in Msh2 À/À cells.…”

“…15 Similarly, we introduced target 1 into Msh3 À/À ES cells. 22 Non-chemically modified deoxyribonucleotides were obtained from Sigma-Genosys Ltd. Procedures for introducing oligonucleotides in ES cells, selection for G418-resistant colonies and identification and purification of modified cells by PCR were essentially as described before.…”

“…Importantly, as Msh3 À/À ES cells have no overt mutator phenotype and Msh3-deficient mice are not cancer prone, we believe that these cells may become the routine target cell for oligonucleotide-mediated gene disruption. 22 However, more experimentation will be required to identify general rules for oligonucleotidemediated base substitution.…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…It should be noted that double-mutant MSH3 −/− MSH6 −/− mice have phenotypes very similar to those of MSH2 −/− (de Wind et al, 1999;. This reflects the fact that MSH2 is a common subunit for both MutSα and MutSβ, the two MutS homologues that carry out MMR on base-base and insertion-deletion mismatches, respectively.…”

“…MSH3 −/− mice do not have a significant phenotype, although a small subset develop gastrointestinal tumours (de Wind et al, 1999;Edelmann et al, 2000). This correlates with the fact that MSH3 mutations are very rare in human HNPCC, and to the fact that the MSH2-MSH6 complex is partially redundant for repair of a single base insertion.…”