Mesalazine reduces mutations in transforming growth factor beta receptor II and activin type II receptor by improvement of replication fidelity in mononucleotide repeats

Abstract

Purpose: Mesalazine (5-aminosalicylic acid, 5-ASA) has chemopreventive properties in colitis-associated cancer. In vitro, it improves replication fidelity at (CA)13 microsatellites independent of mismatch repair proficiency. Therefore, 5-ASA might be advantageous in patients with hereditary nonpolyposis colorectal cancer. At this point, however, it is uncertain whether this improvement of replication fidelity is specific for (CA)13 repetitive sequences. Here, we tested the effect of 5-ASA on replication fidelity in mononucleotide, dinucleotide, and tetranucleotide repeats.

Experimental design: HCT116 and HCT116+chr3 cells were transfected with pIREShyg2-EGFP reporter plasmids harboring the following microsatellites: A10, G10, (CA)13, (CA)26, (AAAG)17, poly-A tracts, and their flanking sequences of transforming growth factor beta receptor II (TGFBR2; A10) and activin type II receptor (ACVR2; A8). Stably transfected single-cell clones were selected, characterized by Southern blotting, sorted into six-well plates, and cultured with or without 5-ASA. Frameshift mutations that shift the enhanced green fluorescence protein into its proper reading frame were quantified by flow cytometry.

Results: In HCT116, 5-ASA reduced the mutant fraction at (CA)13 by 48.3%, at A10 by 35.6-43.6%, at G10 by 74.9-83.6%, and at (AAAG)17 by 37.6-44.4%. Similar results were observed in hMLH1-proficient HCT116+chr3 cells. Moreover, the presence of 5-ASA significantly reduced mutations in TGFBR2 (A10) and ACVR2 (A8) by 39.9% and 46.2%, respectively.

Conclusions: 5-ASA increases replication fidelity in mononucleotide, dinucleotide, and tetranucleotide repeats and reduces mutations in tumor suppressor genes TGFBR2 and ACVR2, a finding that may provoke in vivo studies for the prevention of colorectal cancer in hereditary nonpolyposis colorectal cancer.

Fig. 1

Effect of 5-ASA on the number of cultured cells. To test the effect of 5-ASA (5 mmol/L) on cell growth, stably transfected cell lines were cultured for 8 d and the total cell count was analyzed. Except for LoVo+chr2 cells a reduction in the total cell count was seen for all clones (P < 0.001). One-way ANOVA method was used for statistical analysis; columns, mean of triplicate cultures for each clone; bars, SEM.

Fig. 2

Effect of 5-ASA on dinucleotide microsatellites. The effect of 5-ASA on replication fidelity within the dinucleotide repeat sequences (CA)13 (A and C) and (CA)26 (B) repeats was tested. Nonfluorescent cells (5 × 10³) were sorted into six-well plates and treated with 5 mmol/L 5-ASA for a period of 8 d. The EGFP-positive mutant fraction was analyzed by flow cytometry. Columns, mean of triplicate cultures for each clone; bars, SEM.

Fig. 3

Effect of 5-ASA on mononucleotide and tetranucleotide microsatellites. The effect of 5-ASA on replication fidelity within mononucleotide repeat sequences A10, G10 (A), and (AAAG)17 repeats (B) was tested. Nonfluorescent cells (5 × 10³) were sorted into six-well plates and treated with 5 mmol/L 5-ASA for a period of 8 d. The EGFP-positive mutant fraction was analyzed by flow cytometry. Columns, mean of triplicate cultures for each clone; bars, SEM.

Fig. 4

Effects of 5-ASA on poly-A microsatellites in TGFBR2 and ACVR2. 5-ASA was tested on replication fidelity in A10 and A8 repeats within flanking regions of exon 3 and 10 of TGFBR2 and ACVR2, respectively. Nonfluorescent cells (5 × 10³) were sorted into six-well plates and treated with 5 mmol/L 5-ASA for a period of 8 d. The EGFP-positive mutant fraction was quantified by flow cytometry. Columns, mean of triplicate cultures for each clone; bars, SEM.