DNA damage responses in relation to late effects following radiotherapy for early breast cancer.

Abstract

10626 Background: In this study, we assessed if induction of apoptosis in G0 blood lymphocytes following ex vivo irradiation correlated with clinical radiosensitivity and DNA double-strand (DSB) repair in breast radiotherapy (RT) patients. Using small molecule inhibitors, we examined the relationship between DSB repair and radiation-induced apoptosis. Methods: Breast cancer patients with minimal (controls) or marked late RT changes (cases) were selected. DSB were quantified by γH2AX/53BP1 immunostaining 0.5 and 24 h after exposure of lymphocytes to 0.5 and 4 Gy, respectively. Induction of apoptosis was assessed 48 h after 8 Gy using a fluorochrome inhibitor of caspases (FLICA) assay. Apoptotic cells were defined by small cell size and positive FLICA signal, measured using flow cytometry. Small molecule DNA-PK (Nu7441) and ATM (Ku55933) inhibitors were used at concentrations of 1 µM and 10 µM, respectively. Results: Despite similar foci induction at 0.5 h, residual foci levels 24 h after 4 Gy differed significantly between cases and controls (Foci per cell = 12.7 in cases, n = 8 vs 10.3 in controls, n = 8, p = 0.002). In contrast to residual foci levels, comparable levels of apoptosis were observed between cases and controls 48 h after 8 Gy. Mean proportion of apoptotic cells was 37.2% in cases and 34.7% in controls (p = 0.413). Residual foci levels were not correlated with levels of apoptosis in lymphocytes of the same patients (R = -0.059, p = 0.785). To determine if apoptosis is modulated by DSB repair, apoptosis measurements were repeated in lymphocytes treated with Nu7441 to inhibit non-homologous end-joining. We observed that Nu7441-treated cells had higher levels of residual foci and apoptosis compared to mock-treated cells, 48 h after 1 Gy. This effect was wholly dependent on an active ATM function as cells treated simultaneously with Nu7441 and Ku55933 had extremely low levels of apoptosis, comparable to levels observed in cells treated with Ku55933 alone. Conclusions: Higher levels of residual DSB observed among radiosensitive patients suggest a role for DSB repair in the pathogenesis of late radiation-induced effects. Induction of apoptosis appears to be dependent on DSB end-joining following exposure to ionising radiation.