Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells
Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Metho...
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2013
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03603016_v87_n4_p785_Bracalente http://hdl.handle.net/20.500.12110/paper_03603016_v87_n4_p785_Bracalente |
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paper:paper_03603016_v87_n4_p785_Bracalente2023-06-08T15:34:36Z Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells Molinari de Rey, Beatriz Palmieri, Mónica Alejandra Kreiner, Andrés Juan Davidson, Jorge Durán, Hebe Alicia Catalytic subunits Chinese Hamster ovary cells DNA double-strand breaks DNA-dependent protein kinase Double-strand breaks High linear energy transfers Methods and materials Nonhomologous end joining Cell culture DNA Enzymes Irradiation Phosphorylation Proteins Repair Radiation deoxyribonucleic acid protein kinase histone H2AX lithium protein kinase proton unclassified drug animal cell article cell mutant cell nucleus cell survival CHO cell clonogenic assay controlled study DNA damage DNA end joining repair double stranded DNA break enzyme active site enzyme activity female gamma radiation immunofluorescence ionizing radiation linear energy transfer nonhuman priority journal protein dephosphorylation protein phosphorylation radiation dose radiation response radiosensitivity Animals Biological Markers CHO Cells Cricetinae Cricetulus DNA Breaks, Double-Stranded DNA Repair DNA-Activated Protein Kinase Gamma Rays Histones Linear Energy Transfer Radiation Dosage Radiation Tolerance Time Factors Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm2) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation. © 2013 Elsevier Inc. Fil:Molinari, B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Palmieri, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kreiner, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Davidson, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Durán, H. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2013 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03603016_v87_n4_p785_Bracalente http://hdl.handle.net/20.500.12110/paper_03603016_v87_n4_p785_Bracalente |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Catalytic subunits Chinese Hamster ovary cells DNA double-strand breaks DNA-dependent protein kinase Double-strand breaks High linear energy transfers Methods and materials Nonhomologous end joining Cell culture DNA Enzymes Irradiation Phosphorylation Proteins Repair Radiation deoxyribonucleic acid protein kinase histone H2AX lithium protein kinase proton unclassified drug animal cell article cell mutant cell nucleus cell survival CHO cell clonogenic assay controlled study DNA damage DNA end joining repair double stranded DNA break enzyme active site enzyme activity female gamma radiation immunofluorescence ionizing radiation linear energy transfer nonhuman priority journal protein dephosphorylation protein phosphorylation radiation dose radiation response radiosensitivity Animals Biological Markers CHO Cells Cricetinae Cricetulus DNA Breaks, Double-Stranded DNA Repair DNA-Activated Protein Kinase Gamma Rays Histones Linear Energy Transfer Radiation Dosage Radiation Tolerance Time Factors |
| spellingShingle |
Catalytic subunits Chinese Hamster ovary cells DNA double-strand breaks DNA-dependent protein kinase Double-strand breaks High linear energy transfers Methods and materials Nonhomologous end joining Cell culture DNA Enzymes Irradiation Phosphorylation Proteins Repair Radiation deoxyribonucleic acid protein kinase histone H2AX lithium protein kinase proton unclassified drug animal cell article cell mutant cell nucleus cell survival CHO cell clonogenic assay controlled study DNA damage DNA end joining repair double stranded DNA break enzyme active site enzyme activity female gamma radiation immunofluorescence ionizing radiation linear energy transfer nonhuman priority journal protein dephosphorylation protein phosphorylation radiation dose radiation response radiosensitivity Animals Biological Markers CHO Cells Cricetinae Cricetulus DNA Breaks, Double-Stranded DNA Repair DNA-Activated Protein Kinase Gamma Rays Histones Linear Energy Transfer Radiation Dosage Radiation Tolerance Time Factors Molinari de Rey, Beatriz Palmieri, Mónica Alejandra Kreiner, Andrés Juan Davidson, Jorge Durán, Hebe Alicia Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| topic_facet |
Catalytic subunits Chinese Hamster ovary cells DNA double-strand breaks DNA-dependent protein kinase Double-strand breaks High linear energy transfers Methods and materials Nonhomologous end joining Cell culture DNA Enzymes Irradiation Phosphorylation Proteins Repair Radiation deoxyribonucleic acid protein kinase histone H2AX lithium protein kinase proton unclassified drug animal cell article cell mutant cell nucleus cell survival CHO cell clonogenic assay controlled study DNA damage DNA end joining repair double stranded DNA break enzyme active site enzyme activity female gamma radiation immunofluorescence ionizing radiation linear energy transfer nonhuman priority journal protein dephosphorylation protein phosphorylation radiation dose radiation response radiosensitivity Animals Biological Markers CHO Cells Cricetinae Cricetulus DNA Breaks, Double-Stranded DNA Repair DNA-Activated Protein Kinase Gamma Rays Histones Linear Energy Transfer Radiation Dosage Radiation Tolerance Time Factors |
| description |
Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm2) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation. © 2013 Elsevier Inc. |
| author |
Molinari de Rey, Beatriz Palmieri, Mónica Alejandra Kreiner, Andrés Juan Davidson, Jorge Durán, Hebe Alicia |
| author_facet |
Molinari de Rey, Beatriz Palmieri, Mónica Alejandra Kreiner, Andrés Juan Davidson, Jorge Durán, Hebe Alicia |
| author_sort |
Molinari de Rey, Beatriz |
| title |
Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| title_short |
Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| title_full |
Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| title_fullStr |
Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| title_full_unstemmed |
Induction and persistence of large γh2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells |
| title_sort |
induction and persistence of large γh2ax foci by high linear energy transfer radiation in dna-dependent protein kinase-deficient cells |
| publishDate |
2013 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03603016_v87_n4_p785_Bracalente http://hdl.handle.net/20.500.12110/paper_03603016_v87_n4_p785_Bracalente |
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