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Molecular and Cellular Biology, February 2002, p. 1049-1059, Vol. 22, No. 4
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.4.1049-1059.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation

Bo Xu, Seong-Tae Kim, Dae-Sik Lim,,{dagger} and Michael B. Kastan*

Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Received 25 July 2001/ Returned for modification 23 August 2001/ Accepted 15 November 2001

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 G1, S, and G2 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 G2 checkpoint and a prolonged G2 arrest after IR, suggesting the existence of different types of G2 arrest. Two molecularly distinct G2/M checkpoints were identified, and the critical importance of the choice of G2/M checkpoint assay was demonstrated. The first of these G2/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 G2 at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G2 cells must be used to assess this arrest. In contrast, G2/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. G2/M accumulation after IR is not affected by the early G2/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 G2 arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G2 checkpoints appear to affect survival following irradiation. Thus, two different G2 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.

* Corresponding author. Mailing address: Department of Hematology-Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale St. Memphis, TN 38105. Phone: (901) 495-3968. Fax: (901) 495-3966. E-mail: Michael.Kastan{at}stjude.org.

{dagger} Present address: Graduate School of Life Science and Biotechnology, Korea University, Sungbuk-ku, Seoul 136-701, Korea.

Molecular and Cellular Biology, February 2002, p. 1049-1059, Vol. 22, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.4.1049-1059.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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