Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity

Ihara, Masataka; Suwa, Akira; Komatsu, Kenshi; Shimasaki, Tatsuya; Okaichi, Kumio; Hendrickson, Eric A.; Okumura, Yutaka

doi:10.1080/095530099140717

Cited by 33 publications

(22 citation statements)

References 21 publications

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“…1) Matsumoto et al, 1997;Beck and Dynlacht, 2001). Several investigators have demonstrated that Ku80 loses its activity rapidly after cells are heated, and may be the heat-sensitive component of DNA-PK (Ihara et al, 1999). Despite evidence that the protein becomes altered and loses its activity after heat shock, thus implicating it as a target for heat-inhibition of DSB repair, a role in heat-radiosensitization has yet to be conclusively demonstrated.…”

Section: Discussionmentioning

confidence: 97%

“…It is believed that such alterations result in a binding of the proteins to DNA or the nuclear matrix, resulting in a masking of sites of radiation-induced DNA damage, or a reduction in accessibility of repair proteins to sites of DSBs (Roti Roti and Winward, 1978;Tomasovic et al, 1978;Warters et al, 1987;Warters et al, 1993;Roti Roti et al, 1997). Conversely, studies on DNA polymerases and more recent studies involving DSB repair proteins suggest that denaturation or aggregation may directly result in inactivation or decreased functionality of the DNA repair enzymes themselves that are responsible for DSB repair (Spiro et al, 1982;Jorritsma et al, 1985;Kampinga et al, , 1993Raaphorst et al, 1993;Burgman et al, 1997;Matsumoto et al, 1997;Ihara et al, 1999). Recent results from cell-free studies performed by Wachsberger and Iliakis (2000) also argue against a role for reduction in accessibility of repair proteins in heat-inhibition of DSB repair.…”

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confidence: 92%

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The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

Dynlacht

Bittner

Bethel

et al. 2003

Journal Cellular Physiology

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A synergistic increase in cell killing is observed when a heat-shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat-radiosensitization, is believed to be mediated by inhibition of repair of radiation-induced double strand breaks (DSB) when cells are exposed to temperatures above 42 degrees C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation-induced DSBs are repaired via the non-homologous end-joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA-dependent protein kinase (DNA-PK), a complex involved in NHEJ, appear to be susceptible to a heat-induced loss of DNA-binding activity, with Ku80 representing the heat-sensitive component. Since the heat-induced loss and subsequent recovery of Ku-DNA binding activity correlates well with heat-radiosensitization, a role for Ku80 and NHEJ in heat-radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat-radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5-45.5 degrees C induce a similar amount of aggregation of Ku80 in human U-1 melanoma cells. These data suggest that the time-temperature-dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat-radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80(-/-) mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild-type (WT) cells, we found that heat-radiosensitization was not reduced in the Ku80(-/-) cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat-radiosensitization. Non-involvement of Ku-dependent or Ku-independent NHEJ pathways in heat-radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV-defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation-induced DSBs and as a target for heat-radiosensitization.

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Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 92%

The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

Dynlacht

Bittner

Bethel

et al. 2003

Journal Cellular Physiology

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“…To this purpose, we studied the DNA repair machinery activation induced by ICG photoactivation at 810 nm. To define the nature of cell damage, the activation of Ku70 and Ku80 heterodimer subunits was observed because their expression is involved in the regulation of cell death induction and inhibited by heat shock [23][24][25] . Moreover, the activation of the proapoptotic pathway after damage induced by ICG photosensitization at 810 nm was observed.…”

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confidence: 99%

Modulation of Ku70/80, Clusterin/ApoJ Isoforms and Bax Expression in Indocyanine-Green-Mediated Photo-Oxidative Cell Damage

Ricci¹,

Pucci²,

Sesti³

et al. 2007

Ophthalmic Res

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dium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h. Conclusion: These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.

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“…Several lines of evidence show that factors involved in DSB repair are likely to be affected by heat shock, for example, Ku heterodimer (Ku70/Ku80) [33][34][35][36], MRN complex [37][38][39], breast cancer susceptibility gene 1 (BRCA1) [40], BRCA2 [41] and RAD51 [41,42]. Heat shock affects these proteins by causing denaturation, cytoplasmic translocation from the nucleus, and protein degradation by the proteasome pathway [43].…”

Section: Discussionmentioning

confidence: 99%

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Takahashi

Mori

Nakagawa

et al. 2016

International Journal of Hyperthermia

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Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (cH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G 2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged cH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs. ARTICLE HISTORY

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Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity

Abstract: DNA-PK was inactivated by heat treatment at 44 degrees C. Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK.

Cited by 33 publications

References 21 publications

The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

The non‐homologous end‐joining pathway is not involved in the radiosensitization of mammalian cells by heat shock

Modulation of Ku70/80, Clusterin/ApoJ Isoforms and Bax Expression in Indocyanine-Green-Mediated Photo-Oxidative Cell Damage

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

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