Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR–Chk1 pathway

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Abstract

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR+ human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3 μM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR+ HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM−/− and ATM+ human fibroblasts, suggesting that the ATM–Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR–Chk1 pathway in the MMR+ cells, resulting in a G2/M checkpoint response

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Materials and methods

Cells and cell culture. HeLa cells, a human cervical carcinoma cell line which is known to be MMR-proficient [16], were grown in RPMI 1640 medium supplemented with 10% fetal calf serum in a humidified atmosphere of 10% CO2 and 90% air at 37 °C. Human ATM−/− fibroblast cells (FT169A) and its wild-type ATM transfected cells (ATM+, YZ5) (generous gifts of Dr. Y. Shiloh) [17] were also grown under the conditions described above.

Transfection of siRNA. Transfection was performed with oligofectamine as

Chk1 and Chk2 are phosphorylated during 6-TG treatment

We used an antibody against phosphorylated serine 317 of Chk1 to examine the involvement of the checkpoint kinase Chk1 in HeLa cells following 6-TG treatment. Phosphorylation of serine 317 is essential for the full activation of Chk1 [23]. Phosphorylation of Chk1 was gradually increased in HeLa cells during 4 days of treatment with 3 μM 6-TG (Fig. 1). The extent of Chk1 phosphorylation is temporally correlated with the 6-TG-induced G2/M peak induction (Fig. 2B), similar to our recently published

Discussion

In this study, we demonstrate that the ATR–Chk1 pathway participates in a G2/M cell cycle checkpoint activation by 6-TG-induced DNA mismatches. We show that a reduction of ATR kinase by siRNA strongly inhibits the G2/M checkpoint response and results in an induction of apoptosis after 6-TG treatment (Fig. 2). Our data also show that 6-TG treatment results in a critical phosphorylation of Chk1 (Fig. 1). Consistently, a reduction of Chk1 kinase results in failure of the formation of a major G2/M

Acknowledgements

We thank Mr. Michael Sramkoski and Dr. James Jacobberger of CWRU for flow cytometry techniques. This work was supported by NIH Grant CA84578 (T.J.K., PI), and the flow cytometry core of the Case Comprehensive Cancer Center at University Hospitals of Cleveland/CWRU (P30 CA43703).

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