Suppression of gene amplification and chromosomal DNA integration by the DNA mismatch repair system

Abstract

Mismatch repair (MMR)-deficient cells are shown to produce >15-fold more methotrexate-resistant colonies than MMR normal cells. The increased resistance to methotrexate is primarily due to gene amplification since all the resistant clones contain double-minute chromosomes and increased copy numbers of the DHFR gene. In addition, integration of linearized or retroviral DNAs into chromosomes is also significantly elevated in MMR-deficient cells. These results suggest that in addition to microsatellite instability and homeologous recombination, MMR is also involved in suppression of other genome instabilities such as gene amplification and chromosomal DNA integration.

Figure 1

Suppression of gene amplification in HCT116 (hMLH1^–/–) by transfected chromosome 3 carrying the wild-type hMLH1 gene. (A and B) Selection of MTX resistance in HCT116+Ch2 (hMLH1^–; MMR^–) and HCT116+Ch3 (hMLH1⁺; MMR⁺) cells. Cells (2 × 10⁶) were plated in 100 × 20 mm plates in the presence of MTX (14 × IC₅₀). The plates were incubated at 37°C for 28 days. Colonies were then fixed and stained with methylene blue. This experiment has been repeated four times and essentially the same results were obtained. (C) IC₅₀ determination using the 4-day MTT assay (55). (D) Determination of the DHFR gene copy number in MTX^r HCT116+Ch2 clones. Genomic DNAs were isolated and the same amount of DNAs was digested with EcoRI and analyzed by agarose gel electrophoresis. The amplified DHFR gene was detected by the ³²P-labeled DHFR cDNA probe. Genomic DNA isolated from HCT116+Ch2 was used as the control. All six MTX^r of HCT116+Ch2 clones contained the amplified 1.8 kb DNA fragment.

Figure 2

Suppression of gene amplification in HCT116 (hMLH1^–/–) by transfected cDNA carrying the wild-type hMLH1 gene. (A) Nuclear expression of hMLH1 in HCT116 cells transfected with wild-type hMLH1 cDNA. Immunostaining analysis of HCT116 and HCT116+hMLH1 cells was performed with monoclonal anti-hMLH1 antibody as described in Materials and Methods. The positive staining of hMLH1 was observed as red–brown color only in the nuclei of HCT116+hMLH1 cells under regular microscopy. (B) Suppression of gene amplification in HCT116 (hMLH1^–/–) cells transfected with the wild-type hMLH1 cDNA. Selection of MTX resistance was performed in HCT116 (MMR^–) and HCT116+hMLH1 (MMR⁺) cells. Cells (2 × 10⁶) were plated in 100 × 20 mm plates in the presence of MTX (14 × IC₅₀). The plates were incubated at 37°C for 28 days. Colonies were then fixed and stained with methylene blue. This experiment has been repeated three times and essentially the same results were obtained.

Figure 3

MMR deficiency enhances chromosomal integration of transfected linear DNA. Three pairs of MMR-deficient and MMR-proficient cells were transfected with linearized pTK-hyg DNAs (see Materials and Methods) and then selected with indicated concentrations of hygromycin B. (A) hMLH1^– (HCT116+Ch2) and hMLH1⁺ (HCT116+Ch3) cells. (B) hMSH6^– (DLD-1) and hMSH6⁺ (DLD-1+Ch2) cells. (C) hMSH2^– (HEC59) and hMSH2⁺ (HEC59+Ch2) cells. The results presented are the average of at least three independent experiments.

Figure 4

MMR deficiency increases the frequency of chromosomal integration of retrovirus. Three pairs of MMR-deficient and MMR-proficient cells were infected with amphotropic retrovirus (see Materials and Methods) and then selected with indicated concentrations of puromycin. (A) hMLH1^– (HCT116+Ch2) and hMLH1⁺ (HCT116+Ch3) cells. (B) hMSH6^– (DLD-1) and hMSH6⁺ (DLD-1+Ch2) cells. (C) hMSH2^– (HEC59) and hMSH2⁺ (HEC59+Ch2) cells. The results are the average of four independent experiments.

Figure 5

Homeologous (non-homologous) recombination processes for gene amplification. (A) NHEJ pathway for inverted duplications. (B) A dumbbell model for inverted dimer formation. Two pairs of inverted repeats are imperfect and form homeologous pairing to generate dumbbell-like DNA intermediate. For details see Introduction and Discussion.