The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells

Tkacz-Stachowska, Kinga; Lund-Andersen, Christin; Velissarou, Angeliki; Myklebust, June Helen; Stokke, Trond; Syljuåsen, Randi G.

doi:10.1016/j.radonc.2011.05.060

Cited by 15 publications

(21 citation statements)

References 20 publications

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“…Checkpoint adaptation has been observed in normal MEF or BJ cells exposed to radiation. 13,41 Yeast are also capable of undergoing checkpoint adaptation. 4 In experiments using other genotoxic agents, normal fibroblasts did not acquire micronuclei after exposure to neocarzinostatin, 42 or hydroxyurea.…”

Section: Micronuclei Cause Additional Damage To Dnamentioning

confidence: 99%

“…10,12 Because these cells have undergone mitosis with damaged DNA, it is likely they acquire changes to their genome. 13 One genomic change that characterizes cancer cells is the presence of micronuclei. Micronuclei form from lagging whole chromosomes 14,15 or chromosome fragments 16 that are not incorporated into the main nucleus following anaphase.…”

Section: Introductionmentioning

confidence: 99%

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Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

Lewis

Golsteyn

2016

Cell Cycle

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We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 mM). Nearly 100% of M059K cells were positive for histone gH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone gH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

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Section: Micronuclei Cause Additional Damage To Dnamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

Lewis

Golsteyn

2016

Cell Cycle

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“…Flow cytometry was conducted as described previously (23). For cell-cycle and DNA damage marker analysis, cells were stained with rabbit anti-phospho-H3 and mouse anti-phospho-H2AX (gH2AX) (Upstate, Millipore).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…DNA was stained with Hoechst 33258 (1.5 mg/mL). Where indicated in the figure legends, bar coding with pacific blue (23,24) was used to eliminate variation in antibody staining or variation in the 5-ethynyl-2 0 -deoxyuridine (EdU) Click-iT reaction between individual samples. Cells were stained with phospho-RPA S4/S8 or S33 antibodies, both at 1:500 (Nordic Biosite).…”

Section: Flow Cytometrymentioning

confidence: 99%

The Efficacy of CHK1 Inhibitors Is Not Altered by Hypoxia, but Is Enhanced after Reoxygenation

Hasvold¹,

Tkacz-Stachowska²,

Rofstad³

et al. 2013

Molecular Cancer Therapeutics

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Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia). The influence of less severe hypoxia on the efficacy of CHK1 inhibitors, administered either as single agents or in combination with other treatments, remains to be investigated. Here, we have assayed the effects of the CHK1 inhibitors, AZD7762 and UCN-01, during various hypoxic conditions and after reoxygenation in the absence and presence of ionizing radiation. Treatment with CHK1 inhibitors during acute or prolonged hypoxia (< 0.03%, 0.2%, and 1% O 2 ; 3 h or 20-24 h) gave similar effects on cell survival as treatment with these inhibitors during normoxia. CHK1 inhibitors combined with ionizing radiation showed similar radiosensitization in hypoxic and normoxic cells. However, when the inhibitors were administered after reoxygenation following prolonged hypoxia (< 0.03% and 0.2%; 20-24 h), we observed decreased cell survival and stronger induction of the DNA damage marker, gH2AX, in S-phase cells. This was accompanied by enhanced phosphorylation of the single-stranded DNA-binding replication protein A. These results suggest that the cytotoxic effects of CHK1 inhibitors are enhanced after reoxygenation following prolonged hypoxia, most likely due to the increased replication-associated DNA damage. Combining CHK1 inhibitors with other treatments that cause increased reoxygenation, such as fractionated radiotherapy, might therefore be beneficial. Mol Cancer Ther; 12(5); 705-16. Ó2013 AACR.

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“…These methods enable high throughput analysis of multiple samples while minimizing inter-sample variability due to procedural limitations (sample processing and instrument variation), and reduce reagent consumption. FCB and MCB have been used in large-scale screening experiments for both drug discovery and immunology research 1, 3–6 .…”

Section: Introductionmentioning

confidence: 99%

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining

Thom²,

et al. 2014

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Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

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The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells

Cited by 15 publications

References 20 publications

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

The Efficacy of CHK1 Inhibitors Is Not Altered by Hypoxia, but Is Enhanced after Reoxygenation

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining

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