The 4 R’s of radiation biology. (A): Repair of sublethal DNA damage. DNA double-strand breaks (DSBs) after exposure to ionizing radiation are mainly repaired by NHEJ. NHEJ involves recognition of the DNA DSBs by Ku70/80, recruitment of the histone H2AX to the DNA leasion, phopshorylation of H2AX by ATM, DNA-PKcs, or ATR, and finally rejoining of the strand ends by XRCC4 and Ligase 4. (B): Redistribution. Mammalian cells exhibit different levels of radioresistance during the course of the cell cycle. Cells in the late S-phase are especially resistant and cells in the G2/M-phase are most sensitive to ionizing radiation. During fractionated radiation cells in the G2/M-phase are preferentially killed. The time between two fractions allows resistant cells from the S-phase of the cell cycle to redistribute into phases in which cells are more radiosensitive. (C): Repopulation. Normal and malignant stem cells have the ability to perform asymmetric cell division, which give rise to a daughter stem cell and a committed progenitor cell. In a symmetric cell division in contrast, stem cells divide into two committed progenitor cells or two daughter stem cells. If the latter happens only in 1% of the stem cell divisions, the number of stem cells after 20 cell doublings will be twice as high as the number of committed progenitor cells. This indicates that small changes in the way stem cells divide have huge impact on the organization of a tissue or tumor and are thought to be the mechanism behind accelerated repopulation. (D): Reoxygenation. Tumors contain regions of hypoxia in which cancer cells are thought to be resistant to radiation. During fractionated radiotherapy, these regions are reoxygenated by various mechanisms including reduction of intratumoral pressure and normalization of the vasculature. Reoxygenation between radiation fractions will lead to radiosensitization of previously hypoxic tumor areas and is thought to increase the efficiency of radiation treatment. Abbreviations: CSCs, cancer stem cells; NHEJ, nonhomologous end joining.

Radiation-induced redistribution and accelerated repopulation employs the developmental Notch pathway. (A): In breast cancer, ionizing radiation induces the expression of Notch receptor ligands on the surface of nontumorigenic cells and possibly other nonmalignant stem cells niche cells like, for example, endothelial cells, which are finally depleted by radiation. Activation of Notch signaling in CSCs may than redistribute quiescent CSCs into the cell cycle in a symmetric type of stem cell division and finally cause repopulation of the tumor. (B): In this model system, TGF-β is produced by the mass of the nontumorigenic, radiosensitive cancer cells and activated by radiation. It antagonizes the proliferative effects of Notch, which is activated in CSCs through interaction with their niche. During the course of fractionated irradiation, most of the nontumorigenic cancer cells are killed. This causes TGF-β levels to drop while Notch is still being activated, resulting in increased regrowth rates and thus accelerated repopulation. Abbreviation: CSCs, cancer stem cells.

CSCs and DNA repair. (A): CSCs exhibit less DNA double-strand breaks (DSBs) after exposure to ionizing radiation than nontumorigenic cells. GCL catalyzes the reaction of cysteine and glutamate to form γ-glutamylcysteine in an ATP-dependent step. In a second step, GSS condensates γ-glutamylcysteine and glycine to form glutathione. Breast CSCs were found to express high levels of GCL and GSS. Consequently, most radiation-induced free radicals were scavenged in breast CSCs and ionizing radiation caused only little DNA damage if compared to nontumorigenic cells. Inhibition of glutamate cysteine ligase by buthionine sulfoximine reversed the radioresistant phenotype. (B): CSCs repair DNA DSBs more efficiently than nontumorigenic cells. CSCs in breast and brain cancers hyperphosphorylated the DNA checkpoint kinases Chk1 and Chk2 constitutively and in response to ionizing radiation, thereby removing DNA DSBs more rapidly and more efficiently. Abbreviations: BSO, Buthionine Sulfoximine; CSCs, cancer stem cells; GCL, glutamate cysteine ligase; GSS, glutathione synthetase; GSSG, glutathione disulfide; ROS, reactive oxygen species.

CSCs and tumor hypoxia. Like long-term repopulating hematopoetic stem cells, quiescent CSCs may exist in a nonperivascular hypoxic niche, relatively protected from ionizing radiation. Activated, and thus cycling, CSCs are found in a perivascular niche with confers increased radiation sensitivity and the dependence of CSCs on that niche makes them vulnerable to anti-angiogenic strategies, which target endothelial cells, thereby destroying the CSC niche. Reoxygenation of the hypoxic CSC niche during radiation fractionation redistributes quiescent CSCs as increasing oxygen levels will modify the niche conditions to render those found in pervascular regions and may cause the transition from a quiescent into an activated, proliferative CSC state. Abbreviation: CSCs, cancer stem cells.