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.

Radiation Sensitivity of Human CD34+Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects

“…Different X-ray doses were used to study residual DNA damage and mutagenic effects. A dose of 4 Gy X rays allowed us to obtain a sufficient number of residual foci after 24 h. The choice of the latter radiation dose was based on previously published studies in which the c-H2AX/ 53BP1 foci assay was used to correlate residual DNA damage with clinical radiosensitivity in breast cancer patients (21,47). However, for the assessment of chromosomal radiosensitivity of the different cell types, a 2 Gy Xray dose was used.…”

“…However, to assess the number of residual DNA DSBs, which may mark unrepaired or misrepaired sites, c-H2AX/53BP1 foci were quantified at 24 h after exposure to 4 Gy X rays. The 4 Gy dose was chosen for the DNA repair experiments, since it is often used for the estimation of radiosensitivity after in vitro irradiation of human lymphocytes (21). Again, colocalizing c-H2AX and 53BP1 foci were scored to make sure that DNA DSBs were analyzed (Fig.…”

Radiogenomics: A systems biology approach to understanding genetic risk factors for radiotherapy toxicity?

“…A significant difference between patients with and without late reaction was not confirmed in a case:control study on breast RT patients but here frozen lymphocytes and a smaller dose were used, and apoptosis was detected by forward/sideward scatter (cell size and granularity) (151). A subsequent smaller study (n=16) using fresh lymphocytes from breast RT patients and a fluorescence-labelled inhibitor of caspases (FLICA) to detect apoptosis also found no difference in mean apoptotic fractions (190). Although not all studies were able to show a significant association of lymphocyte apoptosis with normal-tissue reaction, three studies in which associations with late reactions were significant showed a consistent negative correlation between the frequency of apoptosis and the risk of late reaction (194, 212, 213) while increased early cell death correlated with early toxicity (194, 196, 212).…”

DNA damage foci: Meaning and significance

“…More recently, DSB repair measured using DNA damage foci in ex vivo‐ or in vivo‐irradiated peripheral blood lymphocytes has also been proposed as a predictive marker of individual risk of oral mucositis in head and neck cancer radiotherapy patients [Fleckenstein et al, ; Goutham et al, ; Li et al, ]. Similarly γH2AX foci levels have been associated with the risk of acute [Djuzenova et al, ; Mumbrekar et al, ] and late normal tissue reactions in breast cancer radiotherapy patients [Chua et al, ; Henriquez‐Hernandez et al, ; Chua et al, ], for late toxicity in prostate cancer patients [van Oorschot et al, 2014] and for acute radiotherapy toxicities in paediatric cancer patients [Rübe et al, ]. However, γH2AX foci results had no predictive power for late normal toxicity in gynaecological cancer radiotherapy [Werbrouck et al, ] or in prostate brachytherapy [Olive et al, ], and no genetic influence was observed on individual γH2AX signaling/DSB repair capacity in a nonclinical study involving 198 twins [Garm et al, ].…”