Identification of miRNA signatures associated with radiation-induced late lung injury in mice.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-β/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident

Cellular autofluorescence following ionizing radiation.

Cells often autofluoresce in response to UV radiation excitation and this can reflect critical aspects of cellular metabolism. Here we report that many different human and murine cell types respond to ionizing radiation with a striking rise in autofluorescence that is dependent on dose and time. There was a highly reproducible fluorescent shift at various wavelengths, which was mirrored by an equally reproducible rise in the vital intracellular metabolic co-factors FAD and NADH. It appears that mitochondria, metabolism and Ca(2+) homeostasis are important for this to occur as cells without mitochondria or cells unable to alter calcium levels did not behave in this way. We believe these radiation-induced changes are of biological importance and that autofluorescence may even provide us with a tool to monitor radiation responses in the clinic

Regulatory T cells in radiotherapeutic responses.

Radiation therapy (RT) can extend its influence in cancer therapy beyond what can be attributed to in-field cytotoxicity by modulating the immune system. While complex, these systemic effects can help tip the therapeutic balance in favor of treatment success or failure. Engagement of the immune system is generally through recognition of damage-associated molecules expressed or released as a result of tumor and normal tissue radiation damage. This system has evolved to discriminate pathological from physiological forms of cell death by signaling "danger." The multiple mechanisms that can be evoked include a shift toward a pro-inflammatory, pro-oxidant microenvironment that can promote maturation of dendritic cells and, in cancer treatment, the development of effector T cell responses to tumor-associated antigens. Control over these processes is exerted by regulatory T cells (Tregs), suppressor macrophages, and immunosuppressive cytokines that act in consort to maintain tolerance to self, limit tissue damage, and re-establish tissue homeostasis. Unfortunately, by the time RT for cancer is initiated the tumor-host relationship has already been sculpted in favor of tumor growth and against immune-mediated mechanisms for tumor regression. Reversing this situation is a major challenge. However, recent data show that removal of Tregs can tip the balance in favor of the generation of radiation-induced anti-tumor immunity. The clinical challenge is to do so without excessive depletion that might precipitate serious autoimmune reactions and increase the likelihood of normal tissue complications. The selective modulation of Treg biology to maintain immune tolerance and control of normal tissue damage, while releasing the "brakes" on anti-tumor immune responses, is a worthy aim with promise for enhancing the therapeutic benefit of RT for cancer

Mitochondria and calcium homeostasis are important for irradiated cells to change their autofluorescence.

<p><b>A</b>) Overlay histogram comparing background FL-1 fluorescence in untreated U87 and U87Rho(0) cells. U87 and mitochondria-deficient U87Rho(0) were treated with 10 Gy and FACS-analyzed 24 h later for <b>B</b>) FL-1 autofluorescence and <b>C</b>) cell size. <b>D</b>) Radiation dose-dependent changes in FL-1 autofluorescence in irradiated DC2.4 after 0, 2, 5, and 10 Gy correlate with changes in forward scatter (top) and side scatter (bottom). <b>E</b>) DC2.4 cells were irradiated with 0 or 10 Gy in the presence of 1 µM A23187 or 0.1% DMSO or 5%FBS/PBS and analyzed 24 h later for viability by 7-AAD exclusion (top). Viable cells were compared for cell size (bottom) and FL-1 autofluorescence (middle). Data are mean ± s.e.m. of n = 6 *p<0.05.</p

Intracellular FAD, NAD and NADH rise following irradiation.

<p>Cells were irradiated with 10 Gy or left untreated and incubated for 24 h before harvest and FAD or NAD/NADH assay. <b>A</b>) range of FAD concentrations per 1 million DC2.4. cells (n = 3), <b>B</b>) Mean NADH concentration per 1million cells (n = 2±s.d.), <b>C</b>) mean NAD concentration per 1 million cells of n = 2±s.d., <b>D</b>) ratio of mean NADH/NAD per 1 million cells (n = 2±s.d. *p<0.05).</p

Radiation increases autofluorescence in a dose- and time- dependent fashion.

<p>DC2.4 cells were irradiated or not under standard culture conditions and analyzed by FACS. <b>A</b>) Forward-side scatter dot blot and Fl-1 histogram of untreated cells and cells at 24 h after treatment with 10 Gy. <b>B</b>) (left) Overlay histograms of FL-1 fluorescence in 10 Gy-irradiated cells analyzed at 24, 48 and 72 h and (right) overlay histogram of FL-1 fluorescence after 0, 2, 5 and 10 Gy and analyzed at 48 h. <b>C</b>) Mean FL-1 fluorescence intensity normalized to control of n = 2±s.d. in DC2.4 treated with radiation ranging from 0.005 to 0.5 Gy and analyzed at 48 h after exposure. *p<0.05.</p

Many cell types respond to radiation with a rise in autofluorescence.

<p>Various murine and human cell lines and primary mouse peripheral blood cells were irradiated with 10 Gy (or less when indicated) and left under standard culture conditions for 24 h prior to FACS analysis. Autofluorescence was recorded in FL-1 and cell viability assessed according to 7-AAD dye exclusion in FL-3. <b>A</b>) FL-1 histogram overlays for treated and untreated B16-OVA, 3LL and EG.7-OVA cells. <b>B</b>) Percent increase in mean FL-1 fluorescence and percent loss in viability in the irradiated sample as compared to control of n = 1–8±s.e.m.</p

Lipid-conjugated Smac analogues.

A small library of monovalent and bivalent Smac mimics was synthesized based on 2 types of monomers, with general structure NMeAla-Xaa-Pro-BHA (Xaa=Cys or Lys). Position 2 of the compounds was utilized to dimerize both types of monomers employing various bis-reactive linkers, as well as to modify selected compounds with lipids. The resulting library was screened in vitro against metastatic human breast cancer cell line MDA-MB-231, and the two most active compounds selected for in vivo studies. The most active lipid-conjugated analogue M11, showed in vivo activity while administered both subcutaneously and orally. Collectively, our findings suggest that lipidation may be a viable approach in the development of new Smac-based therapeutic leads

Radiation and Inflammation

The immune system has the power to modulate the expression of radiation-induced normal and tumor tissue damage. On the one hand, it can contribute to cancer cure, and on the other hand, it can influence acute and late radiation side effects, which in many ways resemble acute and chronic inflammatory disease states. The way radiation-induced inflammation feeds into adaptive antigen-specific immune responses adds another dimension to the tumor-host cross talk during radiation therapy and to possible radiation-driven autoimmune responses. Understanding how radiation affects inflammation and immunity is therefore critical if we are to effectively manipulate these forces for benefit in radiation oncology treatments

NRF2 Mediates Cellular Resistance to Transformation, Radiation, and Inflammation in Mice

Nuclear factor erythroid 2-related factor 2 (NRF2) is recognized as a master transcription factor that regulates expression of numerous detoxifying and antioxidant cytoprotective genes. In fact, models of NRF2 deficiency indicate roles not only in redox regulation, but also in metabolism, inflammatory/autoimmune disease, cancer, and radioresistancy. Since ionizing radiation (IR) generates reactive oxygen species (ROS), it is not surprising it activates NRF2 pathways. However, unexpectedly, activation is often delayed for many days after the initial ROS burst. Here, we demonstrate that, as assayed by γ-H2AX staining, rapid DNA double strand break (DSB) formation by IR in primary mouse Nrf2–/– MEFs was not affected by loss of NRF2, and neither was DSB repair to any great extent. In spite of this, basal and IR-induced transformation was greatly enhanced, suggesting that NRF2 protects against late IR-induced genomic instability, at least in murine MEFs. Another possible IR- and NRF2-related event that could be altered is inflammation and NRF2 deficiency increased IR-induced NF-κB pro-inflammatory responses mostly late after exposure. The proclivity of NRF2 to restrain inflammation is also reflected in the reprogramming of tumor antigen-specific lymphocyte responses in mice where Nrf2 k.o. switches Th2 responses to Th1 polarity. Delayed NRF2 responses to IR may be critical for the immune transition from prooxidant inflammation to antioxidant healing as well as in driving cellular radioresistance and survival. Targeting NRF2 to reprogram immunity could be of considerable therapeutic benefit in radiation and immunotherapy