Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase.
Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.
Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK.
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Rainey
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Black
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Zachos
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Gillespie
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002645:Chickens; D004249:DNA Damage; D004261:DNA Replication / Q000502:physiology; D004273:DNA, Neoplasm / Q000528:radiation effects*; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology; D016195:G2 Phase / Q000502:physiology*; D018390:Gene Targeting; D006657:Histones / Q000378:metabolism; D007124:Immunoenzyme Techniques; D016393:Lymphoma, B-Cell / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011839:Radiation, Ionizing; D016196:S Phase / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004273:DNA, Neoplasm; D006657:Histones; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016159:Tumor Suppressor Protein p53; C000605869:Chek1 protein, mouse; C578633:Chek2 protein, mouse; C475961:gamma-H2AX protein, mouse