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Involvement of ATM/ATR-p38 MAPK cascade in MNNG induced G1-S arrest
1Department of Pathology, School of Medicine, Zhejiang University, Hangzhou 310031, Zhejiang Province, China.
Aim:
To understand the effect of low concentration of N-methyl-N'-nitro-nitrosoguanidine (MNNG), which is a widely distributed environmental mutagen and carcinogen especially for human gastric cancer, on DNA damage and to study its possible pathway in regulating cell cycle arrest.
Methods:
The DNA damage effect was measured by Comet assay. A specific phospho-(Ser/Thr) ATM/ATR substrate antibody was used to detect the damage sensor by Western blot. p38 kinase activity was measured by direct kinase assay, and immunoprecipitation for the possible connection between ATM/ATR and p38 MAPK. Flow cytometry analysis and p38 MAPK specific inhibitor SB203580 were combined to detect the possible cell cycle arrest by p38 MAPK.
Results:
With the same low concentration MNNG exposure (0.2 microM 2.5 h), Comet assays indicated that strand breaks accumulated, Western blot and kinase assay showed ATM/ATR and p38 kinase activated, immunoprecipitation showed phospho-ATM/ATR substrate antibody combined with both p38 MAPK antibody and phospho-p38 MAPK antibody. p38 MAPK pathway was involved in the G1-S arrest.
Conclusion:
Activation of ATM/ATR by MNNG induced DNA damage leads to activation of p38 MAPK, which involves in the G1 checkpoint in mammalian cells.
Insights
Low concentrations of N-methyl-N'-nitro-nitrosoguanidine (MNNG) cause DNA damage and activate ATM/ATR and p38 MAPK. This pathway is involved in regulating the G1-S cell cycle arrest in mammalian cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Toxicology
Background:
- N-methyl-N'-nitro-nitrosoguanidine (MNNG) is a mutagen and carcinogen linked to gastric cancer.
- Understanding MNNG's effects on DNA and cell cycle regulation is crucial.
Purpose of the Study:
- To investigate the impact of low-concentration MNNG on DNA damage.
- To elucidate the molecular pathway MNNG uses to induce cell cycle arrest.
Main Methods:
- Comet assay for DNA strand breaks.
- Western blot to detect ATM/ATR and p38 MAPK activation.
- Kinase assays and immunoprecipitation to explore pathway connections.
- Flow cytometry and p38 MAPK inhibitor (SB203580) to assess cell cycle arrest.
Main Results:
- Low MNNG (0.2 μM) induced DNA strand breaks.
- ATM/ATR and p38 MAPK were activated by MNNG exposure.
- Evidence suggests a link between ATM/ATR and p38 MAPK activation.
- The p38 MAPK pathway mediated G1-S phase arrest.
Conclusions:
- MNNG-induced DNA damage activates the ATM/ATR pathway.
- Activated ATM/ATR leads to p38 MAPK activation.
- The ATM/ATR-p38 MAPK axis is critical for the G1 cell cycle checkpoint in response to MNNG.