research papers J. Synchrotron Rad. (2014). 21, 801–810 doi:10.1107/S1600577514011424 801 Journal of Synchrotron Radiation ISSN 1600-5775 Received 3 April 2014 Accepted 17 May 2014 # 2014 International Union of Crystallography Spatial and temporal distribution of cH2AX fluorescence in human cell cultures following synchrotron-generated X-ray microbeams: lack of correlation between persistent cH2AX foci and apoptosis Danielle L. Anderson, a Razmik Mirzayans, a,b Bonnie Andrais, b E. Albert Siegbahn, c B. Gino Fallone a,d and Brad Warkentin a,d * a Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada, b Experimental Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada, c Medical Physics, Stockholm University, Box 260, S-17176 Stockholm, Sweden, and d Medial Physics, Cross Cancer Institute, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada. *E-mail: brad.warkentin@albertahealthservices.ca Formation of  H2AX foci (a marker of DNA double-strand breaks), rates of foci clearance and apoptosis were investigated in cultured normal human fibroblasts and p53 wild-type malignant glioma cells after exposure to high-dose synchrotron-generated microbeams. Doses up to 283 Gy were delivered using beam geometries that included a microbeam array (50 mm wide, 400 mm spacing), single microbeams (60–570 mm wide) and a broad beam (32 mm wide). The two cell types exhibited similar trends with respect to the initial formation and time-dependent clearance of  H2AX foci after irradiation. High levels of  H2AX foci persisted as late as 72 h post-irradiation in the majority of cells within cultures of both cell types. Levels of persistent foci after irradiation via the 570 mm microbeam or broad beam were higher when compared with those observed after exposure to the 60 mm microbeam or microbeam array. Despite persistence of  H2AX foci, these irradiation conditions triggered apoptosis in only a small proportion (<5%) of cells within cultures of both cell types. These results contribute to the understanding of the fundamental biological consequences of high-dose microbeam irradiations, and implicate the impor- tance of non-apoptotic responses such as p53-mediated growth arrest (premature senescence). Keywords: microbeam; cH2AX; X-rays; radiation therapy; DNA damage; apoptosis. 1. Introduction Microbeam radiation therapy (MRT) employs synchrotron X-rays shaped into an array of quasi-parallel microbeams to treat solid tumours. MRT differs markedly from conventional therapies in three key aspects: (i) the dose distribution delivered to the tumours is spatially fractionated (microbeams are typically 25–100 mm wide and separated by 100–400 mm), (ii) extremely high doses (hundreds of Gy) are delivered in the microbeam paths in a single high-dose-rate (thousands of Gy s 1 ) fraction, and (iii) the polyenergetic synchrotron X-rays are relatively low in energy ( 50–600 keV) (Slatkin et al., 1992, 1995; Laissue et al. , 1998; Brauer-Krisch et al., 2010). Animal studies have demonstrated that MRT can achieve similar or improved tumour control while resulting in reduced normal tissue toxicity when compared with single-fraction broad-beam irradiations (Laissue et al. , 1998, 2007; Dilmanian et al., 2001, 2002, 2003; Miura et al., 2006; Regnard et al., 2008; Serduc et al., 2008; Bra ¨uer-Krisch et al., 2010; Crosbie et al. , 2010). A better understanding of normal and tumour tissue response to high-dose microbeams would advance the present preclinical state of MRT, and may lead to translational advancements in conventional therapies. The mechanisms underlying the biological responses to high-dose microbeams are complex and not yet fully under- stood. Animal studies have suggested that the differential responses between normal (skin) and cancerous (mammary) tissues might reflect differences at cellular levels in terms of DNA repair and apoptotic cell death (Crosbie et al. , 2010). The  H2AX assay has been instrumental in measuring DNA damage after microbeam irradiation in various mammalian cell types both in vitro and in vivo (Kashino et al. , 2009; Crosbie et al. , 2010; Sprung et al., 2011; Rothkamm et al., 2012). Phosphorylation of the histone H2A variant H2AX on Ser139