The base excision repair enzyme MED1 mediates DNA damage response to antitumor drugs and is associated with mismatch repair system integrity

Abstract

Cytotoxicity of methylating agents is caused mostly by methylation of the O6 position of guanine in DNA to form O6-methylguanine (O6-meG). O6-meG can direct misincorporation of thymine during replication, generating O6-meG:T mismatches. Recognition of these mispairs by the mismatch repair (MMR) system leads to cell cycle arrest and apoptosis. MMR also modulates sensitivity to other antitumor drugs. The base excision repair (BER) enzyme MED1 (also known as MBD4) interacts with the MMR protein MLH1. MED1 was found to exhibit thymine glycosylase activity on O6-meG:T mismatches. To examine the biological significance of this activity, we generated mice with targeted inactivation of the Med1 gene and prepared mouse embryonic fibroblasts (MEF) with different Med1 genotype. Unlike wild-type and heterozygous cultures, Med1-/- MEF failed to undergo G2-M cell cycle arrest and apoptosis upon treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Similar results were obtained with platinum compounds' 5-fluorouracil and irinotecan. As is the case with MMR-defective cells, resistance of Med1-/- MEF to MNNG was due to a tolerance mechanism because DNA damage accumulated but did not elicit checkpoint activation. Interestingly, steady state amounts of several MMR proteins are reduced in Med1-/- MEF, in comparison with Med1+/+ and Med1+/- MEF. We conclude that MED1 has an additional role in DNA damage response to antitumor agents and is associated with integrity of the MMR system. MED1 defects (much like MMR defects) may impair cell cycle arrest and apoptosis induced by DNA damage.

Fig. 1.

MED1 thymine glycosylase activity for O⁶–meG:T mismatches. Double-stranded oligonucleotides, bearing O⁶–meG:T mismatches in a CpG or GpG context and ³²P-labeled at the 3′ end on the bottom strand, were treated with purified recombinant MED1 protein at 37°C. The reactions were then treated with NaOH at 90°C, to cleave the sugar-phosphate backbone at the abasic site, and were separated by PAGE. OG: O⁶–meG. Lanes 1 is a positive control.

Fig. 2.

Production of two novel murine Med1 alleles. (A) The Δ1–3 and Δ2–5 constructs were prepared by replacing exons (black boxes) 1–3 and 2–5, respectively, with a pgk-neo cassette; thicker lines indicate the recombination arms. The Δ1–3 and Δ2–5 alleles were produced in ES cells after homologous recombination. In the wild-type allele, the SpeI (S) restriction sites are located 13 kb upstream and 11 kb downstream of exon 1, respectively. (B) Southern blot analysis of genomic DNA from MEF of different Med1 genotype. After SpeI digestion and hybridization with a 1.5-kb SacI-HindIII probe located 6 kb upstream of exon 1 (panel A), the wild-type, Δ1–3, and Δ2–5 alleles show a 24-, 13-, and 16-kb fragment, respectively. (C) The expression of Med1 was monitored by RT-PCR analysis of MEF RNA by using primers located in exons 1 and 3 (A). No expression was detected in homologous recombinant cells.

Fig. 3.

Med1^-/- MEF are resistant to cytotoxicity of antitumor agents. (A) Detection of apoptotic mono- and oligonucleosomes in MEF with different genotypes treated with increasing doses of MNNG for 48 h. Nucleosome enrichment is computed with respect to vehicle-treated cells. (B) TUNEL assay of Med1^+/+ (a and b) and Med1^-/- (c and d) MEF treated with 10 μg/ml MNNG for 6 h: a and c, DAPI staining; b and d, TUNEL staining. (C–E) Survival analysis (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay) at 3 and 5 days of MEF with different genotypes treated with 20 μM oxaliplatin, 20 μM irinotecan, and 10 μM 5-FU, respectively. (F) Survival analysis (TUNEL assay) of MEF with different Med1 genotype infected with amphotropic ASV at the indicated multiplicity of infection. The negative and positive controls are uninduced (ATR wild type) GM847 human fibroblasts and GM847 cells induced with doxycycline to express a dominant negative ATR mutant (d.n.), respectively.

Fig. 4.

Accumulation of DNA damage in MEF, irrespective of Med1 genotype. Alkaline elution analysis of [³H]thymidine-labeled genomic DNA from MEF of the indicated genotype treated or not with 10 μg/ml MNNG for 1 h. Percent of radioactivity remaining on filter is plotted as a function of elution time. Results are representative of three independent experiments.

Fig. 5.

Tolerance of Med1-deficient cells to alkylation damage. (A) Cell cycle analysis by flow cytometry of Med1^+/+ and Med1^-/- MEF treated with 0, 0.5 and 1 μg/ml MNNG for 48 and 72 h, and stained with propidium iodide. Each profile represents the analysis of 20,000 events. Yellow boxes mark the subG₁ region with the indicated percent of apoptotic cells. (B) Western blot analysis with anti-phospho-p53 Ser-15 antibody of lysates from MEF treated or not with 0.5 μg/ml MNNG for 24–72 h. The asterisk marks a cross-reacting band. (C) Quantification of the anti-phospho-p53 Ser-15 Western blot (B). Relative units of p53 activation are computed with respect to untreated cells at the same time point.

Fig. 6.

MMR protein levels are reduced in Med1-deficient cells by a posttranscriptional mechanism. (A) Western blot analysis of the indicated MMR proteins in MEF with different Med1 genotype. Probing with β-actin and PCNA antibodies revealed approximately equal loading of lysates. Size of the bands in kDa is shown. (B) Analysis of mRNA abundance of the indicated MMR genes in MEF with different Med1 genotype. Exonic primers for RT-PCR were separated by at least one intron. Size of the bands in base pairs is shown. Control lanes (c) are reactions in which cDNA was omitted. The asterisk marks a nonspecific band in Mlh1 reactions. Glyceraldehyde-3 phosphate dehydrogenase (Gapd) levels indicated the use of approximately equal amounts of RNA.