Heat-Induced Aggregation of XRCC5 (Ku80) in Nontolerant and Thermotolerant Cells

Beck, Brian D.; Dynlacht, Joseph R.

doi:10.1667/0033-7587(2001)156[0767:hiaoxk]2.0.co;2

Cited by 25 publications

(23 citation statements)

References 39 publications

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“…However, very few attempts have been made to evaluate the effect of resistance to stress on the repair efficiency. The work of Beck and Dynlacht (2001) focused on extractability of the XRCC5 polypeptide after heating, in thermotolerant as compared to thermosensitive U-1 human cells. The extractability of XRCC5 was higher in thermotolerant cells than in thermosensitive cells.…”

Section: Discussionmentioning

confidence: 99%

Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum

et al. 2006

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Regulation of genetic variation in natural populations is a problem of primary importance to evolutionary biology. In the reported study, the repair efficiency of double strand DNA breaks was compared in six wild barley accessions from Israeli natural populations of H. spontaneum: three from mesic populations (one from Maalot and two from Mount Meron, Upper Galilee) and three from xeric populations (one from Wadi Quilt in the Judean Desert and two from Sede Boqer, in the northern Negev Desert). Pulsed field gel electrophoresis was used to score double-strand breaks of DNA (DSBs) caused by methyl methanesulphonate (MMS) treatment. All six accessions were also tested for heat tolerance: four of these, three xeric and one mesic (from Maalot population), were scored as heat tolerant whereas both accessions from Mount Meron population displayed heat sensitivity. MMS caused a significant increase in the level of DSBs relative to the control in all accessions. The major questions were whether and how the efficiency of DNA repair after mutagenic treatment is affected by the environmental conditions and accession's adaptation to these conditions. Differences were found among the accessions in the repair pattern. Plants of two out of the four heat tolerant accessions did not manage to repair DNA neither at 25 degrees Celsius nor at 37 degrees Celsius. The remaining two heat tolerant accessions significantly repaired the breaks at 37 degrees Celsius, but not at 25 degrees Celsius. By contrast, plants of the two heat susceptible accessions significantly lowered the level of DSBs at 25 degrees Celsius but not at 37 degrees Celsius. Therefore, the accessions that proved capable to repair the induced damages in DNA at one of the two temperatures displayed a pattern that may imply the existence of a negative feedback mechanism in regulation of genetic variation. Such a dependence of DNA integrity on environment and genotype may serve an important factor for maintaining relatively high level of mutability without increasing the genetic load.

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Section: Discussionmentioning

confidence: 99%

Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum

et al. 2006

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“…Preparation of nuclear and cytoplasmic fractions and quantitative Western blotting for determination of heatinduced changes in Ku80 extractability in U-1 cells have been described previously (Beck and Dynlacht, 2001). Briefly, at various times after heating, cells were trypsinized on ice, centrifuged, and the cell pellet washed in phosphate buffered saline.…”

Section: Measurement Of Ku80 Extractability As An Indicator Of Proteimentioning

confidence: 99%

“…We previously reported that when human U-1 melanoma cells are heated at 45.58C and nuclei are isolated immediately thereafter, a dosedependent decrease in detergent extractability of nuclear Ku80 is observed (Beck and Dynlacht, 2001). The increase in Ku80 retained in the nuclear fraction of heated cells is indicative of aggregation of Ku80 within the nucleus.…”

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confidence: 96%

“…While immunofluorescence labeling techniques clearly indicate that Ku80 is predominantly localized to the nucleus, Ku80 is a soluble protein which is easily extracted from nuclei of cells upon treatment with a nonionic detergent, and is isolated with the cytoplasmic protein fraction (Beck and Dynlacht, 2001). When nuclear proteins become denatured and aggregated to each other or to native proteins (especially proteins of the nuclear matrix), the proteins are more difficult to extract when nuclei are isolated using non-ionic detergents Warters et al, 1993;Borrelli et al, 1996;Roti Roti et al, 1998).…”

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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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“…Cells deficient in Ku are defective in DSB rejoining and hypersensitive to IR. Ku80 becomes aggregated 54,55 and loses its activity rapidly after cells are heated 47,48,56,57 , but even before these findings (and indeed, the identification of the role of the Ku70/80 heterodimer in DSB repair), evidence for a role for Ku80 and for the NHEJ pathway itself in heat radiosensitization had been proposed 58 . However, Woudstra et al 59 and Dynlacht et al 55 have concluded that Ku and DNA ligase IV (and thus NHEJ) are not critical for heat radiosensitization and are suggestive that other pathways for DSB repair, notably HR, may be a target for radiosensitization.…”

Section: Introductionmentioning

confidence: 99%

Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells

Dynlacht

Pandita

et al. 2004

International Journal of Hyperthermia

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The mechanism by which hyperthermia sensitizes mammalian cells to ionizing radiation remains to be elucidated, but an overwhelming amount of circumstantial evidence suggests that heat radiosensitization might be mediated by inhibition of double-strand break repair, particularly after exposure of irradiated cells to heat treatments in excess of about 43 degrees C. In mammalian cells, double-strand break repair usually occurs via two pathways, non-homologous end-joining and homologous recombination. Several reports suggest a role for non-homologous end-joining in heat radiosensitization, while others implicate homologous recombination as a target. However, cell lines that are compromised in either the non-homologous end-joining or homologous recombination pathway are still capable of being radiosensitized, suggesting that heat affects both pathways. Indeed, several of the proteins involved in one or both of these pathways have been observed to undergo alterations or translocation after unirradiated or irradiated cells are exposed to heat shock. The work summarized in this review implicates proteins of the Mre11/Rad50/Nbs1 complex as targets for heat radiosensitization.

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Heat-Induced Aggregation of XRCC5 (Ku80) in Nontolerant and Thermotolerant Cells

Cited by 25 publications

References 39 publications

Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum

Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum

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

Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells

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