Tissue-specific DNA-PK-dependent H2AX phosphorylation and γ-H2AX elimination after X-irradiation in vivo

Koike, Manabu; Sugasawa, Jun; Yasuda, Mariko; Koike, Aki

doi:10.1016/j.bbrc.2008.08.095

Cited by 33 publications

(25 citation statements)

References 14 publications

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“…Moreover, we revealed that in TD-astrocytes, DNA-PK is actually responsible for DNA-damage-induced H2AX phosphorylation, unlike in NSC, where ATM appears to be the main responsible kinase. Such a substituting role of DNA-PK in cells lacking robust ATM activity is consistent with observations in ATM-deficient cells [33][34][35] and mouse models, 36,37 in which DNA-PK can take over the role of ATM in phosphorylating H2AX. It is worth considering that ATM cannot be directly recruited to DSB, rather requiring the DNAbinding role of proteins of the MRE11/RAD50/NBS1 complex, 1 which we found strongly downregulated in astrocytes.…”

Section: Discussionsupporting

confidence: 87%

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

2011

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The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes -the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for cH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR.

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Section: Discussionsupporting

confidence: 87%

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

2011

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“…In line with this hypothesis, accumulation of DNA-PKcs, Ku70 and Ku80 following hypoxia and iron chelation have been demonstrated in a number of different cell lines (Ginis and Faller , 2000 ;Lynch et al , 2001 ;Um et al , 2004 ;Bouquet et al , 2011 ). DNA-PK has been shown to phosphorylate H2AX in different cell lines and in vivo in response to DNA damage (Stiff et al , 2004 ;Koike et al , 2008 ;An et al , 2010 ) under hypertonic conditions (Reitsema et al , 2005 ) and during apoptotic DNA fragmentation (Mukherjee et al , 2006 ). Of note, the hypoxic DNA-PK activation resulted in increased HIF-dependent gene expression (Bouquet et al , 2011 ).…”

Section: Discussionmentioning

confidence: 78%

HIF mediated and DNA damage independent histone H2AX phosphorylation in chronic hypoxia

Wrann

Kaufmann²,

Wirthner³

et al. 2013

Biological Chemistry

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Abstract:The histone variant 2AX (H2AX) is phosphorylated at Serine 139 by the PI3K-like kinase family members ATM, ATR and DNA-PK. Genotoxic stress, such as tumor radioand chemotherapy, is considered to be the main inducer of phosphorylated H2AX ( γ H2AX), which forms distinct foci at sites of DNA damage where DNA repair factors accumulate. γ H2AX accumulation under severe hypoxic/anoxic (0.02 % oxygen) conditions has recently been reported to follow replication fork stalling in the absence of detectable DNA damage. In this study, we found HIF-dependent accumulation of γ H2AX in several cancer cell lines and mouse embryonic fibroblasts exposed to physiologically relevant chronic hypoxia (0.2 % oxygen), which did not induce detectable levels of DNA strand breaks. The hypoxic accumulation of γ H2AX was delayed by the RNAi-mediated knockdown of HIF-1 α or HIF-2 α and further decreased when both HIF-α s were absent. Conversely, basal phosphorylation of H2AX was increased in cells with constitutively stabilized HIF-2 α . These results suggest that both HIF-1 and HIF-2 are involved in γ H2AX accumulation by tumor hypoxia, which might increase a cancer cell ' s capacity to repair DNA damage, contributing to tumor therapy resistance.

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“…Interestingly, addition of a DNA-PKcs inhibitor to SN38 treatment caused an increase in Olive tail moment and was associated with a slight decrease in γH2AX. This conflicting result indicates that phosphorylation of H2AX, a DNA-PKcs substrate [41][42][43][44][45], is partially inhibited in the presence of ICC. Thus, when DNA-PKcs is inhibited, H2AX phosphorylation may not be a true indicator of DNA damage.…”

Section: Dna-pkcs Inhibitors Act Synergistically With Sn38 In Colon Cmentioning

confidence: 78%

Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells

et al. 2011

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This study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC(50) values of SN38 were observed at 5 and 10 μM of DNA-PK inhibitors. Moreover, at 1-2 μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

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Tissue-specific DNA-PK-dependent H2AX phosphorylation and γ-H2AX elimination after X-irradiation in vivo

Cited by 33 publications

References 14 publications

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

HIF mediated and DNA damage independent histone H2AX phosphorylation in chronic hypoxia

Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells

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