title: Hyperthermia does not affect rejoining of DNA double-strand breaks in a cell-free assay.
abstract: OBJECTIVE
  Heat radiosensitization is poorly understood but is believed to be caused by an inhibition in the repair of radiation-induced DNA lesions. This inhibition in DNA repair may be caused either by direct heat inactivation of repair enzymes, or by heat-induced protein denaturation that leads to their precipitation onto nuclear chromatin structures, generating a barrier that prevents repair enzymes from reaching the damage sites.
  
  
  METHODS
  A previously described (Ganguly and Iliakis, Int J Radiat Biol 1995, 68, 447-457) cell-free assay was introduced to evaluate rejoining of radiation-induced DNA double-strand breaks (dsb) in heated (45.5 degrees C, 20 min) nuclei prepared from A549 cells, in reactions assembled with extracts of non-heated and non-irradiated HeLa cells. The assay allowed the functional evaluation of the effect of precipitated nuclear protein on dsb rejoining. By combining heated nuclei with extracts of non-heated cells the assay avoided complications that would otherwise arise when intact cells are studied, where both nuclear structures and repair factors are heated and therefore potentially altered.
  
  
  RESULTS
  It was observed that exposure of A549 cells to 45.5 degrees C for 20 min caused a 50% increase in the relative protein content of isolated nuclei but had no effect on the in vitro rejoining of dsb. In agreement with earlier reports, a greatly reduced rate of dsb rejoining was observed either in intact A549 or HeLa cells after exposure to heat.
  
  
  CONCLUSIONS
  The results indicate that an increased retention of proteins in heated nuclei is not necessarily associated with an inhibition of dsb rejoining. While the in vitro system may only reproduce certain aspects of the in vivo conditions, the results suggest that protein accretion as a mechanism of heat radiosensitization requires further testing using functional assays.
authors.affiliation: Department of Radiation Oncology, Kimmel Cancer Center of Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Wachsberger
authors.forename: G
authors.initials: G
authors.lastname: Iliakis
mesh_terms: D002457:Cell Extracts / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism; D019457:Chromosome Breakage / Q000235:genetics*; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology*; D004307:Dose-Response Relationship, Radiation; D006367:HeLa Cells / Q000502:physiology / Q000528:radiation effects; D006358:Hot Temperature; D006801:Humans; D014176:Protein Biosynthesis; D014407:Tumor Cells, Cultured
chemical_list: D002457:Cell Extracts; D004247:DNA