title: DNA double-strand break repair and induction of apoptosis in ex vivo irradiated blood lymphocytes in relation to late normal tissue reactions following breast radiotherapy.
abstract: This study aimed to test whether induction of apoptosis following ex vivo X-irradiation of unstimulated blood lymphocytes correlated with clinical radiosensitivity and DNA double-strand break (DSB) repair in breast radiotherapy patients and healthy volunteers. Using small molecule inhibitors, the relationship between DSB repair and radiation-induced apoptosis was examined. Sixteen breast cancer patients with minimal (controls, n = 8) or extremely marked late radiation-induced change (cases, n = 8) and eight healthy volunteers were selected. DSBs were quantified by γH2AX/53BP1 immunofluorescence, and apoptosis was measured using a fluorogenic inhibitor of caspases assay. Mean γH2AX/53BP1 focus levels 24 h after exposure to 4 Gy were higher in cases (12.7 foci per cell) than in controls (10.3 foci per cell, p = 0.002). In contrast, the mean apoptotic fraction 48 h after 8 Gy was comparable, 37.2 % in cases and 34.7 % in controls (p = 0.442). Residual focus and apoptosis levels were not correlated within individuals (Spearman's R = -0.0059, p = 0.785). However, cells treated with DNA-PK inhibitor Nu7441 had higher focus and apoptosis levels 48 h after 1 Gy compared to mock-treated cells, suggesting that apoptosis induction following irradiation is modulated by DSB repair. This effect required functional ATM since cells treated simultaneously with Nu7441 and the ATM inhibitor Ku55933 were resistant to apoptosis despite high levels of residual foci. One clinical case displayed an impaired DNA-PK-dependent end-joining cellular phenotype. In summary, clinical radiosensitivity may be associated with impaired DSB repair in some patients. Although pharmaceutical inhibition of ATM and DNA-PK affected apoptosis induction and DSB repair, no association was observed between apoptosis and residual focus levels in patients and volunteers.
authors.affiliation: Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, OX11 0RQ, UK.
authors.forename: Melvin Lee Kiang
authors.initials: ML
authors.lastname: Chua
authors.forename: Simon
authors.initials: S
authors.lastname: Horn
authors.forename: Navita
authors.initials: N
authors.lastname: Somaiah
authors.forename: Sue
authors.initials: S
authors.lastname: Davies
authors.forename: Lone
authors.initials: L
authors.lastname: Gothard
authors.forename: Roger
authors.initials: R
authors.lastname: A'Hern
authors.forename: John
authors.initials: J
authors.lastname: Yarnold
authors.forename: Kai
authors.initials: K
authors.lastname: Rothkamm
mesh_terms: D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D064007:Ataxia Telangiectasia Mutated Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001943:Breast Neoplasms / Q000532:radiotherapy*; D016022:Case-Control Studies; D002867:Chromones / Q000494:pharmacology; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects*; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects*; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D008875:Middle Aged; D009025:Morpholines / Q000494:pharmacology; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D058958:Organs at Risk / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology; D011832:Radiation Injuries / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D011836:Radiation Tolerance / Q000187:drug effects / Q000528:radiation effects; D013997:Time Factors; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: C495818:2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; C499693:8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one; D002867:Chromones; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011753:Pyrones; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human