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

TY - JOUR

T1 - 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

AU - Chua, Melvin Lee Kiang

AU - Horn, Simon

AU - Somaiah, Navita

AU - Davies, Sue

AU - Gothard, Lone

AU - A'Hern, Roger

AU - Yarnold, John

AU - Rothkamm, Kai

PY - 2014/5

Y1 - 2014/5

N2 - 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.

AB - 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.

KW - Adult

KW - Aged

KW - Apoptosis/drug effects

KW - Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors

KW - Breast Neoplasms/radiotherapy

KW - Case-Control Studies

KW - Chromones/pharmacology

KW - DNA Breaks, Double-Stranded/drug effects

KW - DNA Repair/drug effects

KW - DNA-Activated Protein Kinase/antagonists & inhibitors

KW - Enzyme Activation/drug effects

KW - Female

KW - Histones/metabolism

KW - Humans

KW - Intracellular Signaling Peptides and Proteins/metabolism

KW - Lymphocytes/cytology

KW - Middle Aged

KW - Morpholines/pharmacology

KW - Nuclear Proteins/antagonists & inhibitors

KW - Organs at Risk/radiation effects

KW - Protein Kinase Inhibitors/pharmacology

KW - Pyrones/pharmacology

KW - Radiation Injuries/genetics

KW - Radiation Tolerance/drug effects

KW - Time Factors

KW - Tumor Suppressor p53-Binding Protein 1

U2 - 10.1007/s00411-014-0531-z

DO - 10.1007/s00411-014-0531-z

M3 - SCORING: Journal article

C2 - 24622963

VL - 53

SP - 355

EP - 364

JO - RADIAT ENVIRON BIOPH

JF - RADIAT ENVIRON BIOPH

SN - 0301-634X

IS - 2

ER -