br When DNA damage occurs cellular repair mechanisms known a
When DNA damage occurs, cellular repair mechanisms known as DNA damage responses (DDRs) are triggered to detect the damage lo-cation, signal their presence, and repair the damaged DNA. H2AX is a histone protein randomly incorporated into the nucleosome—the basic repeat unit of chromatin—during replication . It is a substrate of several protein kinases related to phosphorinositide-3-kinase, such as ataxia telangiectasia mutated, ataxia telangiectasia and Rad3–related, and DNA-dependent protein kinase . In DDRs, phosphorylation of H2AX is crucial for DNA DSB repair initiation. Phosphorylated H2AX, or γ-H2AX, mediates DNA DSB repair protein recruitment and regula-tion [12,13]. The accumulation and dissolution of γ-H2AX determine the extent of DNA DSB sensing and repair.
2. Materials and methods
2.1. Cell irradiation system
The National Tsing Hua University (NTHU) cell irradiation system was designed and constructed by Cho et al. in 2013 . The irradia-tion platform, established on the beam line of a 3-MV Van de Graaff accelerator at the NTHU accelerator laboratory, is able to deliver ver-tical homogeneous α-particle or proton beams for cell irradiation. A diagram of the NTHU cell irradiation system is presented in Fig. 1. In the main scattering chamber, a 100-nm-thin gold film tilted at 45° is used as the particle scattering foil. A 1-mm-diameter beam exit aperture opens directly above the scattering foil and is sealed with a 100-nm Si3N4 membrane to maintain a vacuum in the main scattering chamber. The Van de Graaff accelerator’s charged-particle beam, focused and shaped by a series of quadrupole magnets and collimators, is guided into the main scattering chamber. There, the incident charged particles are scattered by the gold foil, and only those particles that are vertically scattered can escape through the exit aperture for cell irradiation. This system also features a beam Menaquinone-4 chamber behind the scattering chamber to absorb unused charged particles and suppress the particle-induced photon background. Through this design, the NTHU cell irra-diation system can provide a uniform, vertically directed proton or α-particle beam. The maximum particle energy is 2.5 MeV, and the beam flux can be controlled at 200–500 particles per second in the beam aperture position. Moreover, a specially designed silicon nitride base culture dish was used in this study to avoid unwanted α-particle energy loss before reaching the target cell. The base of this culture dish was formed by a 15 × 15 × 0.35 mm3 silicon chip, on which two 1 × 1 mm2 apertures were made and covered by a 100-nm silicon
nitride membrane. Our earlier research already demonstrated that a silicon nitride membrane has suitable biocompatibility and attachment yield for the adherent cell. However, because the silicon chip thickness was larger than the α-particle range, only those cells seeding in the vicinity of the apertures could be α-particle irradiated. A detailed de-scription of the cell irradiation system and silicon nitride base cell culture dish may be found in our earlier studies [14,15]. In this study, general GBM cells and GBM CSCs were seeded on the silicon nitride base culture dish separately. Both cell lines were irradiated with 2.0-MeV α-particles at 1.6 Gy, and γ-H2AX expression was observed at 0.5, 1, 4, 12, and 24 h after irradiation.
The permanent human brain malignant glioma cell line 8401 (GBM 8401), established from a 31-year-old Chinese woman with GBM, was kindly provided by Dr. Wei-Hwa Lee. In Dr. Lee’s laboratory, GBM 8401 was subcultured in vitro in a monolayer culture for more than 100 passages over 24 months. A study by Lee et al. revealed the tumor-igenicity of the GBM 8401 cell line in athymic nude mice . In the present study, the GBM 8401 cell line was cultured in an RPMI 1640 medium, with 10% fetal bovine serum and 1% penicillin–streptomycin solution added to provide sufficient nutrients for cell growth. Unlike the general GBM cell line, isolated GBM CSCs were cultivated in an RPMI 1640 medium without serum, to which B-27 supplement (17504; Gib-co, Waltham, USA), N2 supplement (17502; Gibco, Waltham, USA), fibroblast growth factor (F0291; Sigma-Aldrich, St. Louis, USA), and epidermal growth factor (E9644; Sigma-Aldrich, St. Louis, USA) were added to provide special nutrients and growth factors for stem cell cultivation.
Because CSCs are a minor subpopulation within general tumor cells, they necessarily have certain similarities with such cells. Therefore, finding specific methods or markers to isolate CSCs from general tumor cells is critical to CSC research. Four methods have been well verified and widely used to isolate CSCs from tumors, namely, CSC-specific cell surface marker identification, side population phenotype detection through Hoechst 33342 exclusion, floating spheres growth capability, and aldehyde dehydrogenase activity . In this study, we employed CSC-specific cell surface marker identification to isolate CSCs from the general GBM cell line. As the target protein, we chose CD133, a transmembrane glycoprotein widely expressed in various organs. Other studies have designated CD133 as an essential marker for identifying CSCs in leukemia, brain tumors, prostate cancer, hepatoma, pancreatic cancer, and colon cancer [18,19]. Lathia et al. reported that CD133+ GBM CSCs are capable of self-renewal through symmetric and asym-metric cell division .