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


METHODS
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.
Campregher
Christoph
C
Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Medical University of Vienna, Vienna, Austria.
Honeder
Clemens
C
Chung
Heekyung
H
Carethers
John M
JM
Gasche
Christoph
C
D030301:Activin Receptors, Type II / Q000235:genetics*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D015139:Blotting, Southern; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology / Q000517:prevention & control; D004261:DNA Replication / Q000187:drug effects*; D005434:Flow Cytometry; D016368:Frameshift Mutation / Q000235:genetics*; D006801:Humans; D019804:Mesalamine / Q000494:pharmacology*; D018895:Microsatellite Repeats / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000894:Anti-Inflammatory Agents, Non-Steroidal; D018125:Receptors, Transforming Growth Factor beta; D019804:Mesalamine; D017346:Protein Serine-Threonine Kinases; D030301:Activin Receptors, Type II; D000077294:Receptor, Transforming Growth Factor-beta Type II; C425585:activin receptor type II-A