Methylation-induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1

Abstract

The mismatch repair (MMR) gene hMLH1 is mutated in approximately 50% of hereditary non-polyposis colon cancers and transcriptionally silenced in approximately 25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR-proficient and arrested at the G(2)/M cell cycle checkpoint following treatment with the DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR-proficient cells, activation of the MNNG-induced G(2)/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

Fig. 1. Inducible hMLH1 expression in 293T Lα cells. (A) In the Tet-Off system, hMLH1 is expressed in the absence of Dox, because the tTA factor binds to the promoter of the expression vector and thus activates transcription. Addition of Dox to the culture medium causes a conformational change in tTA, which leads to its dissociation from the promoter and thus to an inactivation of hMLH1 transcription. (B) Western blot analysis of cytoplasmic (CE) and nuclear (NE) extracts of cells cultured in the absence (–) or presence (+) of 50 ng/ml Dox. hMLH1 and hPMS2 were visualized using anti-hMLH1 or anti-hPMS2 antibodies as described in Materials and methods. Total extract (TE) of MMR proficient HeLa cells was used as a positive control. (C) Stability of hMutLα. The cells were cultured without Dox (–) to induce maximal hMLH1 expression. Following the addition of 50 ng/ml Dox (+), total cell extracts were isolated after 1, 2, 3, 4, 6 and 8 days. Western blot analysis was performed using anti-hMLH1 and anti-hPMS2 antibodies as in (B).

Fig. 2. MMR proficiency of 293T Lα cell extracts. (A) Repair efficiency of a G/T mismatch in the M13mp2 vector carrying a strand discrimination signal 3′ from the mispair, using cytoplasmic extracts of the 293T Lα⁺ and 293T Lα^– cells, supplemented or not with recombinant hMutLα (see text for details). Error bars show standard errors. (B) Correction of a G/T mismatch within a BglII restriction site of a pGEM vector, following incubation with nuclear extracts of 293T Lα⁺ or 293T Lα^– cells, supplemented or not with recombinant hMutLα. The strand discrimination signal in this heteroduplex substrate was 5′ from the mispair. Efficient repair resulted in the restoration of a BglII site and in the generation of two DNA fragments that co-migrate with those observed in the reference digest of the homoduplex molecule carrying a bona fide BglII site.

Fig. 3. Sensitivity of 293T Lα cells to MNNG. (A) Survival of 293T Lα⁺ and 293T Lα^– cells following treatment with 5 µM MNNG. 293 and 293T cells were used as MMR-proficient and -deficient controls, respectively. The presence of Dox (+Dox) in the culture medium did not affect the control cells, but had a dramatic effect on the survival of the 293T Lα cell populations. (B) IC₅₀ values of 293T Lα⁺ and 293T Lα^– cells. Each value represents the mean ± SE. (C) Cell cycle profiles of 293T Lα⁺ and 293T Lα^– cells treated with 0.2 µM MNNG. Shown are representative cytometrograms of cells expressing (293T Lα⁺) and not expressing (293T Lα^–) hMLH1. G₁, cell population in the G₁ phase of the cell cycle with a 2n DNA content; G₂, cells in the G₂ and M stages of the cell cycle with a 4n DNA content; S, cells in various stages of DNA synthesis with a DNA content between 2n and 4n.

Fig. 4. Post-translational protein modification and strand break processing in MNNG-treated 293T Lα cells. (A) Phosphorylation status of p53 and cdc2 in 293T Lα⁺ and 293T Lα^– cells 1–4 days after treatment with 0.2 µM MNNG. P-p53, P-cdc2, phosphorylated p53 and cdc2 proteins, respectively; C, untreated control cells; β-tubulin, internal standard used to ascertain equal gel loading. (B) γ-H2AX foci formation in MNNG-treated 293T Lα cells. In the control cell population, <10% of cells displayed H2AX foci. Following MNNG treatment, all cells contained foci until 24 h post-treatment. See text for details and Materials and methods for experimental procedures.

Fig. 4. Post-translational protein modification and strand break processing in MNNG-treated 293T Lα cells. (A) Phosphorylation status of p53 and cdc2 in 293T Lα⁺ and 293T Lα^– cells 1–4 days after treatment with 0.2 µM MNNG. P-p53, P-cdc2, phosphorylated p53 and cdc2 proteins, respectively; C, untreated control cells; β-tubulin, internal standard used to ascertain equal gel loading. (B) γ-H2AX foci formation in MNNG-treated 293T Lα cells. In the control cell population, <10% of cells displayed H2AX foci. Following MNNG treatment, all cells contained foci until 24 h post-treatment. See text for details and Materials and methods for experimental procedures.

Fig. 5. Mismatch correction efficiency and MNNG-induced G₂/M arrest in cells expressing different amounts of hMLH1. (A) Dependence of hMLH1 expression on Dox concentration. hMLH1 and hPMS2 were visualized as described in Materials and methods. β-tubulin, internal standard used to ascertain equal loading. (B) MMR efficiency of a G/T mispair in an M13mp2 substrate carrying a strand-discrimination signal 3′ from the mispair. Error bars show standard errors. (C) Variation in doubling times of 293T Lα cells grown in the indicated Dox concentrations following treatment with 5 µM MNNG. (D) FACS analysis of 293T Lα cell populations grown in the indicated Dox concentrations, either untreated (Control), or 72 h after treatment with 0.2 µM MNNG (see also Figure 3C). (E) Phosphorylation of p53 and cdc2 48 h after treatment of cells (grown in the indicated Dox concentrations) with 0.2 µM MNNG. β-tubulin, internal standard used to ascertain equal loading.