scholarly journals Artemis Links ATM to G2/M Checkpoint Recovery via Regulation of Cdk1-Cyclin B

2007 ◽  
Vol 27 (7) ◽  
pp. 2625-2635 ◽  
Author(s):  
Liyi Geng ◽  
Xiaoshan Zhang ◽  
Shu Zheng ◽  
Randy J. Legerski
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Checkpoint ◽  
Nonhomologous End Joining ◽  
Cyclin B ◽  
End Joining ◽  
M Cell ◽  
Strand Breaks ◽  
Checkpoint Recovery ◽  
Cycle Checkpoint

ABSTRACT Artemis is a phospho-protein that has been shown to have roles in V(D)J recombination, nonhomologous end-joining of double-strand breaks, and regulation of the DNA damage-induced G2/M cell cycle checkpoint. Here, we have identified four sites in Artemis that are phosphorylated in response to ionizing radiation (IR) and show that ATM is the major kinase responsible for these modifications. Two of the sites, S534 and S538, show rapid phosphorylation and dephosphorylation, and the other two sites, S516 and S645, exhibit rapid and prolonged phosphorylation. Mutation of both of these latter two residues results in defective recovery from the G2/M cell cycle checkpoint. This defective recovery is due to promotion by mutant Artemis of an enhanced interaction between unphosphorylated cyclin B and Cdk1, which in turn promotes inhibitory phosphorylation of Cdk1 by the Wee1 kinase. In addition, we show that mutant Artemis prevents Cdk1-cyclin B activation by causing its retention in the centrosome and inhibition of its nuclear import during prophase. These findings show that ATM regulates G2/M checkpoint recovery through inhibitory phosphorylations of Artemis that occur soon after DNA damage, thus setting a molecular switch that, hours later upon completion of DNA repair, allows activation of the Cdk1-cyclin B complex. These findings thus establish a novel function of Artemis as a regulator of the cell cycle in response to DNA damage.

scholarly journals Artemis Is a Phosphorylation Target of ATM and ATR and Is Involved in the G2/M DNA Damage Checkpoint Response

2004 ◽  
Vol 24 (20) ◽  
pp. 9207-9220 ◽  
Author(s):  
Xiaoshan Zhang ◽  
Janice Succi ◽  
Zhaohui Feng ◽  
Sheela Prithivirajsingh ◽  
Michael D. Story ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Severe Combined Immunodeficiency ◽  
Cell Cycle Checkpoint ◽  
End Joining ◽  
M Cell ◽  
Checkpoint Kinase ◽  
Checkpoint Response ◽  
Cell Extracts ◽  
Cycle Checkpoint

ABSTRACT Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

scholarly journals Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint

2006 ◽  
Vol 25 (4) ◽  
pp. 763-773 ◽  
Author(s):  
Gustavo Pedraza-Alva ◽  
Miroslav Koulnis ◽  
Colette Charland ◽  
Tina Thornton ◽  
James L Clements ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Map Kinase ◽  
P38 Map Kinase ◽  
Cell Cycle Checkpoint ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
M Cell ◽  
Strand Breaks ◽  
Cycle Checkpoint

scholarly journals Activation of Akt/Protein Kinase B Overcomes a G2/M Cell Cycle Checkpoint Induced by DNA Damage

2002 ◽  
Vol 22 (22) ◽  
pp. 7831-7841 ◽  
Author(s):  
Eugene S. Kandel ◽  
Jennifer Skeen ◽  
Nathan Majewski ◽  
Antonio Di Cristofano ◽  
Pier Paolo Pandolfi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Es Cells ◽  
Cell Cycle Checkpoint ◽  
Wild Type ◽  
M Cell ◽  
Human Cancers ◽  
Cycle Checkpoint

ABSTRACT Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G2/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G2/M to the G1 phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G2 arrest that is alleviated by activated Akt. Furthermore, the transition from the G2/M to the G1 phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G2/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G2/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.

The Wee1 Inhibitor MK1775 In Combination With Cytarabine (AraC) Has Potent Activity In AML By Completely Abrogating DNA Damage and Cell Cycle Checkpoint Repair Capacity Via The Mrn/NBS1 Complex

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3831-3831
Author(s):  
Leena Chaudhuri ◽  
James M Bogenberger ◽  
Lisa Sproat ◽  
James L Slack ◽  
Veena Fauble ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Single Agent ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Leukemic Cells ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Mitotic Entry ◽  
Cycle Checkpoint

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Saccharomyces cerevisiae as a Model System To Define the Chromosomal Instability Phenotype

2005 ◽  
Vol 25 (16) ◽  
pp. 7226-7238 ◽  
Author(s):  
Christopher D. Putnam ◽  
Vincent Pennaneach ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Genome Instability ◽  
Human Cancer ◽  
Terminal Segment ◽  
Cell Cycle Checkpoint ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Cycle Checkpoint ◽  
Chromosome Fusions

ABSTRACT Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.

scholarly journals 53BP1 is required for class switch recombination

2004 ◽  
Vol 165 (4) ◽  
pp. 459-464 ◽  
Author(s):  
Irene M. Ward ◽  
Bernardo Reina-San-Martin ◽  
Alexandru Olaru ◽  
Kay Minn ◽  
Koji Tamada ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Cell Cycle Checkpoint ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Checkpoint Control ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Cycle Checkpoint

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for “classic” nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

scholarly journals WEE1 Inhibition Enhances Anti-Apoptotic Dependency as a Result of Premature Mitotic Entry and DNA Damage

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1743 ◽  
Author(s):  
Mathilde Rikje Willemijn de Jong ◽  
Myra Langendonk ◽  
Bart Reitsma ◽  
Pien Herbers ◽  
Marcel Nijland ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell Lymphoma ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Checkpoints ◽  
Specific Cell ◽  
M Cell ◽  
Mitotic Entry ◽  
Enhanced Sensitivity ◽  
Cycle Checkpoint

Genomically unstable cancers are dependent on specific cell cycle checkpoints to maintain viability and prevent apoptosis. The cell cycle checkpoint protein WEE1 is highly expressed in genomically unstable cancers, including diffuse large B-cell lymphoma (DLBCL). Although WEE1 inhibition effectively induces apoptosis in cancer cells, the effect of WEE1 inhibition on anti-apoptotic dependency is not well understood. We show that inhibition of WEE1 by AZD1775 induces DNA damage and pre-mitotic entry in DLBCL, thereby enhancing dependency on BCL-2 and/or MCL-1. Combining AZD1775 with anti-apoptotic inhibitors such as venetoclax (BCL-2i) or S63845 (MCL-1i) enhanced sensitivity in a cell-specific manner. In addition, we demonstrate that both G2/M cell cycle arrest and DNA damage induction put a similar stress on DLBCL cells, thereby enhancing anti-apoptotic dependency. Therefore, genotoxic or cell cycle disrupting agents combined with specific anti-apoptotic inhibitors may be very effective in genomic unstable cancers such as DLBCL and therefore warrants further clinical evaluation.

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