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Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation
Bo Xu1, Seong-Tae Kim, Dae-Sik Lim
1Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Abstract:
Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G(1), S, and G(2) phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G(2) checkpoint and a prolonged G(2) arrest after IR, suggesting the existence of different types of G(2) arrest. Two molecularly distinct G(2)/M checkpoints were identified, and the critical importance of the choice of G(2)/M checkpoint assay was demonstrated. The first of these G(2)/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G(2) at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G(2) cells must be used to assess this arrest. In contrast, G(2)/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G(2)/M accumulation after IR is not affected by the early G(2)/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G(2) arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G(2) checkpoints appear to affect survival following irradiation. Thus, two different G(2) arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.
Insights
Ionizing radiation triggers two distinct G(2) cell cycle arrests in mammalian cells. Understanding these ATM-dependent and ATM-independent pathways is crucial for accurate cell cycle checkpoint analysis.
Area of Science:
- Cell Biology
- Genomics
- Cancer Research
Background:
- Cell cycle checkpoints are vital for maintaining genomic integrity and preventing cancer.
- Ionizing radiation (IR) induces cell cycle arrests, with the ATM protein playing a key role in G(1), S, and G(2) phases.
- ATM-deficient cells show defective G(2) checkpoints and prolonged G(2) arrest, suggesting distinct G(2) arrest mechanisms.
Purpose of the Study:
- To identify and characterize distinct G(2)/M checkpoints following IR.
- To highlight the importance of selecting appropriate cell cycle assays for studying these checkpoints.
- To elucidate the roles of ATM, Nbs1, and Brca1 in G(2) arrest mechanisms.
Main Methods:
- Analysis of cell cycle progression after IR using assays that distinguish G(2) and mitotic phases.
- Comparison of IR-induced G(2) arrest in wild-type and ATM-deficient cells.
- Investigation of G(2)/M accumulation using propidium iodide staining.
- Assessment of IR effects on cells lacking S-phase checkpoint components (Nbs1, Brca1).
Main Results:
- Two distinct G(2)/M checkpoints were identified: an early, transient, ATM-dependent checkpoint and a later, prolonged, ATM-independent accumulation.
- The early G(2) arrest prevents cells in G(2) at IR exposure from entering mitosis.
- The later G(2)/M accumulation involves cells from earlier phases and is enhanced in cells with defective S-phase checkpoints.
Conclusions:
- Mammalian cells possess at least two distinct G(2) arrest mechanisms in response to IR.
- The choice of cell cycle assay is critical for accurately assessing G(2) arrest and checkpoint function.
- Neither identified G(2) checkpoint significantly impacts cell survival post-irradiation.