DNA Protein Kinase–Dependent G2 Checkpoint Revealed following Knockdown of Ataxia-Telangiectasia Mutated in Human Mammary Epithelial Cells

Arlander, Sonnet J.H.; Greene, Bryan T.; Innes, Cynthia L.; Paules, Richard S.

doi:10.1158/0008-5472.can-07-0675

Cited by 31 publications

(36 citation statements)

References 48 publications

(60 reference statements)

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“…Moreover, although blocking the Chk2 mobility shift they did not abolish T68 phosphorylation, indicating that ATM is dispensable for this modification. This conclusion is supported by the observations in ATMdeficient HMEC (Arlander et al, 2008) or NHF (Tomimatsu et al, 2009). On the other hand, KU-55933 and caffeine had a limited effect on the ATR pathway, as they did not block Chk1 activation (Figure 7d).…”

Section: Resultssupporting

confidence: 78%

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

Lossaint

Besnard²,

Fisher³

et al. 2011

Oncogene

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In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/ Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant.Here we compared the respective roles of Chk1, Chk2 and p21 in DNA damage-induced G2 arrest in normal human fibroblasts, normal epithelial cells and frequently used p53 proficient cancer cells. We show that in normal cells, Chk1, but not Chk2, is involved in G2 arrest whereas neither are essential. In contrast, p21 is required. However, Chk1, but not Chk2, becomes necessary for arrest in U2OS osteosarcoma cells. We find that their ATM/p53/p21 response in G2 phase is defective, like in other cancer cells with wild-type p53, and conclude that cross-talk between the Chk1 and p21 pathways allows them to switch dependency for G2 arrest onto Chk1. Using the specific ATM inhibitor KU-55933 we confirm the essential role of ATM in the induction of p21 for G2 arrest of normal cells. Efficient p21 induction is required for nuclear sequestration of inactive cyclin B1-Cdk1 complexes preceding irreversible cell cycle exit in G2. Our results demonstrate that p21 is able to fulfill the Chk1 functions in G2 arrest under continuous genotoxic stress, which has important implications for cancer chemotherapy.

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Section: Resultssupporting

confidence: 78%

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

Lossaint

Besnard²,

Fisher³

et al. 2011

Oncogene

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“…In contrast, DNA-PKcs has been identified as an important enzyme in the NHEJ of DNA double-strand break repair and in triggering double-strand break-induced apoptosis. A recent report showed its function on the G 2 checkpoint in response to IR (30). In this study, we have shown another important role of DNA-PKcs in maintaining the stability of spindle formation and negatively regulating mitotic catastrophe in response to IR.…”

Section: Discussionsupporting

confidence: 60%

“…DNA-PKcs is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand breaks (28), V(D)J recombination of immunoglobulin genes and T-cell receptor genes (27), and telomere length maintenance (29). More recently, DNA-PKcs has been shown to contribute to the G 2 checkpoint in response to IR (30). DNA-PKcs has also been shown to phophorylate a number of Ataxia-telangiectasia mutated kinase (ATM) substrates in DNA damage response, e.g., KRAB-associated protein 1 and H2AX (31).…”

Section: Introductionmentioning

confidence: 99%

Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage

Shang

Huang

et al. 2010

Cancer Research

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DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Here, we showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by small interfering RNA or specific inhibitor NU7026 resulted in an increased outcome of polyploidy after 2-Gy or 4-Gy irradiation. Simultaneously, a high incidence of multinucleated cells and multipolar spindles was detected in DNA-PKcs-deficient cells. Time-lapse video microscopy revealed that depression of DNA-PKcs results in mitotic catastrophe associated with mitotic progression failure in response to DNA damage. Moreover, DNA-PKcs inhibition led to a prolonged G 2 -M arrest and increased the outcome of aberrant spindles and mitotic catastrophe in Ataxia-telangiectasia mutated kinase (ATM)-deficient AT5BIVA cells. We have also revealed the localizations of phosphorylated DNA-PKcs/T2609 at the centrosomes, kinetochores, and midbody during mitosis. We have found that the association of DNA-PKcs and checkpoint kinase 2 (Chk2) is driven by Ku70/80 heterodimer. Inactivation of DNA-PKcs strikingly attenuated the ionizing radiation-induced phosphorylation of Chk2/T68 in both ATMefficient and ATM-deficient cells. Chk2/p-T68 was also shown to localize at the centrosomes and midbody. These results reveal an important role of DNA-PKcs on stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage and provide another prospect for understanding the mechanism coupling DNA repair and the regulation of mitotic progression. Cancer Res; 70(9); 3657-66. ©2010 AACR.

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“…Importantly, although ATM-deficient cells do not maintain a G 1 -phase cell cycle arrest upon induction of DSBs, ATM-independent G 2 /M checkpoint arrest responses to genotoxic stressors (including doxorubicin) do occur (47)(48)(49)(50)(51). As expected, culture of the ATM-deficient (AT) human fibroblast line GM05849 with doxorubicin for 24 h did not, in contrast to WT cells, arrest them in G 1 phase (Fig.…”

Section: Doxorubicin-induced Up-regulation Of Cyclin G2 Is Atmindepensupporting

confidence: 54%

“…Although ATR and ATM both enforce the DSB DDR delay in M-phase entry, ATR activity is thought to regulate the majority of the late (2-9 h post ␥-irradiation) phase of the checkpoint response (4,46,65). In the absence of ATM, both ATR and DNA-PK regulate DNA DSB G 2 /M checkpoint responses (50,51). We showed (Fig.…”

Section: Discussionmentioning

confidence: 72%

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

Zimmermann

Arachchige-Don

Donaldson

et al. 2012

Journal of Biological Chemistry

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Background: DNA damage triggers cell cycle checkpoints to halt cell division ahead of DNA repair. Results: Ectopic cyclin G2 (CycG2) induces a Chk2-dependent cell cycle arrest, and depletion of endogenous CycG2 attenuates doxorubicin-induced G 2 /M-phase cell cycle arrest. Conclusion: CycG2 influences checkpoint signaling and is required for G 2 /M arrest responses to genotoxic stress. Significance: Proper checkpoint function is important for genomic integrity and tumor suppression.

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DNA Protein Kinase–Dependent G2 Checkpoint Revealed following Knockdown of Ataxia-Telangiectasia Mutated in Human Mammary Epithelial Cells

Abstract: Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA

Cited by 31 publications

References 48 publications

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

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