DNA-dependent protein kinase regulates cytosolic double-stranded DNA secretion from irradiated macrophages to increase radiosensitivity of tumors.

BACKGROUND AND PURPOSE: To investigate the molecular mechanism by which irradiated macrophages secrete cytosolic double-stranded DNA (c-dsDNA) to increase radiosensitivity of tumors. MATERIALS AND METHODS: Irradiated bone marrow-derived macrophages (BMDM) were co-incubated with irradiated EO771 or MC38 cancer cells to determine clonogenic survival. c-dsDNA were measured by agarose gel or enzyme-linked immunosorbent assay. BMDM or cancer cells were analyzed with immunostaining or western blot. Subcutaneously implanted MC38 cells in myeloid-specific Prkdc knockout (KO) mice or littermate control mice were irradiated with 8 Gy to determine radiosensitivity of tumors. RESULTS: We observed that irradiated BMDM significantly increased radiosensitivity of cancer cells. By performing immunostaining, we found that there was a dose-dependent increase in the formation of c-dsDNA and phosphorylation in DNA-dependent protein kinase (DNA-PK) in irradiated BMDM. Importantly, c-dsDNA in irradiated BMDM could be secreted to the extracellular milieu and this process required DNA-PK, which phosphorylated myosin light chain to regulate the secretion. The secreted c-dsDNA from irradiated BMDM then activated toll-like receptor-9 and subsequent nuclear factor kappa-light-chain-enhancer of activated B cells signaling in the adjacent cancer cells inhibiting radiation-induced DNA double strand break repair. Lastly, we observed that irradiated tumors in vivo had a significantly increased number of tumor-associated macrophages (TAM) with phosphorylated DNA-PK expression in the cytosol. Furthermore, tumors grown in myeloid-specific Prkdc KO mice, in which TAM lacked phosphorylated DNA-PK expression were significantly more radioresistant than those of the wild-type control mice. CONCLUSIONS: Irradiated macrophages can increase antitumor efficacy of radiotherapy through secretion of c-dsDNA under the regulation of DNA-PK.

FLASH Radiotherapy: A FLASHing Idea to Preserve Neurocognitive Function.

FLASH radiotherapy (FLASH RT) is a technique to deliver ultra-high dose rate in a fraction of a second. Evidence from experimental animal models suggest that FLASH RT spares various normal tissues including the lung, gastrointestinal track, and brain from radiation-induced toxicity (a phenomenon known as FLASH effect), which is otherwise commonly observed with conventional dose rate RT. However, it is not simply the ultra-high dose rate alone that brings the FLASH effect. Multiple parameters such as instantaneous dose rate, pulse size, pulse repetition frequency, and the total duration of exposure all need to be carefully optimized simultaneously. Furthermore it is critical to validate FLASH effects in an in vivo experimental model system. The exact molecular mechanism responsible for this FLASH effect is not yet understood although a number of hypotheses have been proposed including oxygen depletion and less reactive oxygen species (ROS) production by FLASH RT, and enhanced ability of normal tissues to handle ROS and labile iron pool compared to tumors. In this review, we briefly overview the process of ionization event and history of radiotherapy and fractionation of ionizing radiation. We also highlight some of the latest FLASH RT reviews and results with a special interest to neurocognitive protection in rodent model with whole brain irradiation. Lastly we discuss some of the issues remain to be answered with FLASH RT including undefined molecular mechanism, lack of standardized parameters, low penetration depth for electron beam, and tumor hypoxia still being a major hurdle for local control. Nevertheless, researchers are close to having all answers to the issues that we have raised, hence we believe that advancement of FLASH RT will be made more quickly than one can anticipate.

Sensing the oxygen and temperature in the adipose tissues - who's sensing what?

Adipose tissues, composed of various cell types, including adipocytes, endothelial cells, neurons, and immune cells, are organs that are exposed to dynamic environmental challenges. During diet-induced obesity, white adipose tissues experience hypoxia due to adipocyte hypertrophy and dysfunctional vasculature. Under these conditions, cells in white adipose tissues activate hypoxia-inducible factor (HIF), a transcription factor that activates signaling pathways involved in metabolism, angiogenesis, and survival/apoptosis to adapt to such an environment. Exposure to cold or activation of the ß-adrenergic receptor (through catecholamines or chemicals) leads to heat generation, mainly in brown adipose tissues through activating uncoupling protein 1 (UCP1), a proton uncoupler in the inner membrane of the mitochondria. White adipose tissues can undergo a similar process under this condition, a phenomenon known as 'browning' of white adipose tissues or 'beige adipocytes'. While UCP1 expression has largely been confined to adipocytes, HIF can be expressed in many types of cells. To dissect the role of HIF in specific types of cells during diet-induced obesity, researchers have generated tissue-specific knockout (KO) mice targeting HIF pathways, and many studies have commonly revealed that intact HIF-1 signaling in adipocytes and adipose tissue macrophages exacerbates tissue inflammation and insulin resistance. In this review, we highlight some of the key findings obtained from these transgenic mice, including Ucp1 KO mice and other models targeting the HIF pathway in adipocytes, macrophages, or endothelial cells, to decipher their roles in diet-induced obesity.

DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs): Beyond the DNA Double-Strand Break Repair.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase related kinase family is a well-known player in repairing DNA double strand break through non-homologous end joining pathway. This mechanism has allowed us to understand its critical role in T and B cell development through V(D)J recombination and class switch recombination, respectively. We have also learned that the defects in these mechanisms lead to severely combined immunodeficiency (SCID). Here we highlight some of the latest evidence where DNA-PKcs has been shown to localize not only in the nucleus but also in the cytoplasm, phosphorylating various proteins involved in cellular metabolism and cytokine production. While it is an exciting time to unveil novel functions of DNA-PKcs, one should carefully choose experimental models to study DNA-PKcs as the experimental evidence has been shown to differ between cells of defective DNA-PKcs and those of DNA-PKcs knockout. Moreover, while there are several DNA-PK inhibitors currently being evaluated in the clinical trials in attempt to increase the efficacy of radiotherapy or chemotherapy, multiple functions and subcellular localization of DNA-PKcs in various types of cells may further complicate the effects at the cellular and organismal level.