Gastroenterology

Gastroenterology

Volume 132, Issue 1, January 2007, Pages 221-235
Gastroenterology

Basic–alimentary tract
5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

https://doi.org/10.1053/j.gastro.2006.10.016Get rights and content

Background & Aims: 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, HCT116p53−/−, 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.

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Cell Lines

HT29 (mutant p53R273H), LoVo (hMSH2 mutant), and HCT116 (hMLH1 mutant) colorectal cancer cells were obtained from the ATCC. HCT116p53−/− cells (p53 null) were originated in Prof. Burt Vogelstein’s laboratory as described.24 HCT116+chr3 (hMLH1 wild-type) were a kind gift from Dr. Boland’s laboratory.25 Cells were grown in monolayers in Iscove’s modified Dulbecco’s medium (IMDM; GIBCO/Invitrogen, Lofer, Austria) containing 2 nmol/L glutamine and 10% fetal bovine serum (FBS) at 5% CO2. The medium

5-ASA Reduces the Proliferation of Human Colon Cancer Cell Lines

The effect of 5-ASA on cellular viability and cellular growth was evaluated by an assay based on the activity of mitochondrial dehydrogenase, which cleaves a soluble yellow tetrazolium salt (MTT) to blue formazan crystals, a conversion that occurs only in living cells. HCT116, LoVo, and HT29 cells were treated with 5-ASA (0–20 mmol/L) for 48 hours. 5-ASA treatment induced a significant concentration-dependent reduction in the proliferation of all cell lines (Figure 1).

5-ASA Affects Cell Cycle Progression in CRC Cells

We aimed to establish

Discussion

The convincing clinical evidence for chemopreventive effect of 5-ASA in patients with IBD has prompted studies of its molecular mechanisms of action. It is generally believed that 5-ASA has specific activities other than anti-inflammatory or oxygen scavenging that interferes with carcinogenesis.

Our group has previously shown that 5-ASA decreases the rate of spontaneous mutations in colon cancer cells, which was independent from the presence of a functional mismatch repair complex,23 thus

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    Supported by the Austrian Science Fund (grants M-874-B14 to M.G.L. and P18270 to C.G.); by the International Organization of Inflammatory Bowel Diseases; and by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

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