title: The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. jdynlach@iupui.edu
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Dynlacht
authors.forename: M Eric
authors.initials: ME
authors.lastname: Bittner
authors.forename: Jody A
authors.initials: JA
authors.lastname: Bethel
authors.forename: Brian D
authors.initials: BD
authors.lastname: Beck
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D016923:Cell Death / Q000528:radiation effects; D002460:Cell Line; D004249:DNA Damage / Q000528:radiation effects*; D004265:DNA Helicases; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000172:deficiency / Q000235:genetics; D004260:DNA Repair / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D011485:Protein Binding; D011839:Radiation, Ionizing; D011995:Recombination, Genetic / Q000528:radiation effects*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP