5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint.
OBJECTIVE
Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability.


METHODS
CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed.


RESULTS
We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase.


CONCLUSIONS
Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.
Luciani
M Gloria
MG
Medical University of Vienna, Department of Internal Medicine IV, Division of Gastroenterology and Hepatology, Währinger Gürtel 18, A-1090 Vienna, Austria.
Campregher
Christoph
C
Fortune
John M
JM
Kunkel
Thomas A
TA
Gasche
Christoph
C
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D015179:Colorectal Neoplasms / Q000473:pathology / Q000517:prevention & control*; D053843:DNA Mismatch Repair / Q000187:drug effects; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005434:Flow Cytometry; D019073:HT29 Cells; D006801:Humans; D019804:Mesalamine / Q000494:pharmacology*; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D008938:Mitosis / Q000187:drug effects; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000894:Anti-Inflammatory Agents, Non-Steroidal; C113571:CDC45 protein, human; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C113575:Rad17 protein, human; D016159:Tumor Suppressor Protein p53; D019804:Mesalamine; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D004258:DNA Polymerase III; D000070957:MutL Protein Homolog 1; C084859:MCM2 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7