O6-methylguanine-induced cell death involves exonuclease 1 as well as DNA mismatch recognition in vivo

Abstract

Alkylation-induced O(6)-methylguanine (O(6)MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O(6)MeG-DNA methyltransferase (Mgmt) protein. O(6)MeG pairs with T during DNA replication, and if the O(6)MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O(6)MeG:T mismatch detection by MutSalpha and MutLalpha leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O(6)MeG-dependent apoptosis, we used an Mgmt-null mouse model combined with either the Msh6-null mutant (defective in mismatch recognition) or the Exo1-null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt-null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt-null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt-null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O(6)MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt-null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O(6)MeGs.

Fig. 1.

Exo1 is required for alkylation resistance in Mgmt-null MEFs and BM myeloid progenitors. (A) Survival of MEFs exposed to MNNG. Wild-type MEFs are represented by solid squares. Mgmt-null MEFs are as filled inverted triangles, Msh6-null as open circles, and Exo1-null as open diamonds. Mgmt/Exo1-double null MEFs are shown as filled diamonds, and Mgmt/Msh6-double null MEFs are shown as filled circles. The surviving cells were detected 4 days after MNNG treatment. SEM is presented for three or more independent experiments. (B) UV survival of primary low passage MEFs. The genotypes are as listed in A. (C) Survival of BM cells exposed to MNU. Genotypes are as listed in A. (D) Survival of BM cells exposed to MMC. Genotypes are as listed in A. Three mice were used for Mgmt/Exo1 and Mgmt/Msh6-double null animals, whereas two mice were used for all other genotypes.

Fig. 2.

Exo1 deletion in Mgmt-null animals extends whole-animal survival after MNU administration. (A) Kaplan–Meyer plots of survival. Mgmt-null animals (n = 6) are represented in blue, Mgmt/Exo1-double null animals (n = 6) in red. All other genotypes represented in black survived the 30-day toxicity study period. Animals were: wild type (n = 5), Mgmt/Msh6-double null (n = 6), Exo1 (n = 7), and Msh6 (n = 6) single mutants. (B) Representative BM (H&E-stained) in femurs of animals treated with 30 mg/kg 8 days after injection. (Magnification: 100×.)

Fig. 3.

Analysis of thymus from mice treated with 4 mg/kg MNU. (A) (Upper) Representative gross anatomy of thymuses taken from treated mice 8 days after injection at the time of necropsy. (Lower) Representative H&E pictures. (Magnification: 100×.) Histopathological analysis and extent of thymic damage are presented below the H&E pictures. (B) Scatter plot of thymus weights adjusted to percentage body weight of each mouse (11- 15 mice per treatment per genotype). P values denote statistically significant differences between mean thymus weights.

Fig. 4.

Analysis of spleens from mice treated with 4 mg/kg MNU. (A) (Upper) Representative, gross anatomy of spleens taken from treated mice 8 days after treatment. (Lower) Representative H&E pictures. (Magnification: 100×.) Histopathological analysis and extent of splenic damage are presented below the H&E pictures. (B) Scatter plot of spleen weights adjusted to percentage body weight (11–15 mice per treatment per genotype). P values denote statistically significant differences between mean spleen weights.