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Supplementary Data from High-Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones

Interferons (IFNs), including IFN-α/β and IFN-γ, are multifaceted cytokines critical for antitumor immunity but contribute to T cell exhaustion through immune checkpoints. Recently, tumor cell expression of IRF1 was shown to enhance antitumor immunity, but a comprehensive evaluation of how IRF1 regulates IFN responses in the tumor and host has not been performed. Here, we show that Irf1-/- mice display enhanced tumor growth of WT and Irf1-/- tumors, as they fail to recruit sufficient NK cells and cytotoxic T cell (CTL). However, Irf1-/- tumors in wild-type mice displayed rapid immunogenic control, expansion of intratumoral lymphoid cells with enhanced effector programs and diminished exhaustion programs when compared to WT tumors. Mechanistically, IRF1 positively regulates the expression of distinct subsets of anti-tumor immunity suppression genes including the CD274/PD-L1, TRAIL, and IDO-1 checkpoints but not the IFN-inducible chemokines CXCL9-11. Surprisingly, control of Irf1-/- tumors required host IFN-α/β signaling but not IFN-γ signaling, while PD-L1 overexpression only weakly restored tumor growth. Thus, IRF1 selectively regulates the effects of IFNs on tumor cells and the tumor microenvironment and may be targeted to boost anti-tumor immunity. Citation Format: Prabhat Kumar Purbey, Manash K. Paul, Keisuke S. Iwamoto, Allison Daly, Joowon Seo, Ameya S. Champhekar, Katie Campbell, Dorthe Schaue, William H. McBride, Steven M. Dubinett, Antoni Ribas, Stephen T. Smale, Philip O. Scumpia. IRF1 displays opposing tumor cell-intrinsic and -extrinsic roles in anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2325.

The University of California possesses a large number of Cs irradiators that are used in a wide variety of medical and research applications. The university president made a system-wide decision to reduce the potential threat of malevolent use of Cs by switching wherever feasible to x-ray irradiators over a 3-y period of time. A Radioactive Source Replacement Working Group of involved faculty was formed to study the topic and to make recommendations as to when alternative technologies could offer equivalency. The Working Group concluded that x-ray irradiators could replace Cs irradiators in most applications, with some likely exceptions. They found that the depth dose curve for the 320 kVp x-ray irradiator was found to be nearly identical to that of Cs down to a depth in tissue of 4 cm. It was concluded that x rays (energies ≤320 keV) are more biologically effective than Cs gamma rays, suggesting that lower doses of x rays will be required to achieve the same biological endpoint as Cs gamma rays. A simple conversion factor for equating x-ray effects to Cs effects was not recommended because relative biological effectiveness depends on multiple factors. They concluded that each experiment should be individually calibrated when converting from Cs irradiators to x-ray irradiators. The lessons learned from implementing the project to date have shown the importance of having senior management buy-in, involving the research community in the decision making process and allowing for exceptions where equivalency of Cs to x ray cannot be established.

Purpose Second malignancies (SMs) after radiation therapy are rare but serious sequelae of treatment. This study investigates whether radiation therapy use is associated with changes in baseline SM risk. Methods and Materials We extracted all patients with cancer, with or without SM, in the Surveillance, Epidemiology, and End Results database from 1973 to 2014. Cumulative incidence of SM for patients stratified by radiation therapy status was calculated using a competing risk model, both for the entire cohort and for subgroups based on the primary tumor's anatomic location. Results We identified 2,872,063 patients with cancer, including 761,289 patients who received radiation therapy and 2,110,774 who did not. The SM rate at 20 years for patients receiving radiation therapy versus no radiation therapy was 21.4% versus 18.8%. The relative risk for SM associated with radiation therapy for the overall group was 1.138 at 20 years. The relative risks for SM associated with radiation therapy to malignancies arising from central nervous system and orbits, head and neck, thorax, abdomen, and pelvis at 20 years were 0.704, 1.011, 0.559, 0.646, and 1.106 for men and 0.792, 1.298, 1.265, 0.780, and 0.988 for women, respectively. Conclusions The association between SM and radiation therapy varies with both sex and disease anatomic location, with the largest increase in SM seen in females irradiated to the head and neck region. Overall, the absolute change in SM rates associated with radiation therapy remains small, with differences in various clinical contexts.

Purpose The clinical efficacy of radiation therapy is mechanistically linked to ionization-induced free radicals that cause cell and tissue injury through direct and indirect mechanisms. Free radical reaction dynamics are influenced by many factors and can be manipulated by static weak magnetic fields (WMF) that perturb singlet-triplet state interconversion. Our study exploits this phenomenon to directly increase ionizing radiation (IR) dose absorption in tumors by combining WMF with radiotherapy as a new and effective method to improve treatment. Methods and Materials Coils were custom made to produce both homogeneous and gradient magnetic fields. The gradient coil enabled simultaneous in vitro assessment of free radical/reactive oxygen species (ROS) reactivity across multiple field strengths from 6 to 66 G. First, increases in ionizing radiation-induced free radical concentrations using oxidant-sensitive fluorescent dyes in a cell-free system were measured and verified. Next, human and murine cancer cell lines were evaluated in in vitro and in vivo models following exposure to clinically relevant doses of ionizing radiation in combination with WMF. Results Cellular responses to IR and WMF were field strength- and cell line-dependent. WMF was able to enhance IR effects on ROS formation, DNA double strand break formation, cell death, and tumor growth. Conclusions We demonstrate that the external presence of a magnetic field enhances radiation-induced cancer cell injury and death in vitro and in vivo. The effect extends beyond the timeframe when free radicals are induced in the presence of radiation into the window when endogenous free radicals are produced and therefore extends the applicability of this novel adjunct to cancer therapy in the context of radiation treatment.

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.

Background and purpose Second malignancies (SM) after external beam radiotherapy (EBRT) or brachytherapy (BT) for prostate cancer (PCa) are rare but serious sequelae. Materials and methods The Surveillance, Epidemiology, and End Results (SEER) database was used to identify men diagnosed with cT1-2N0M0 PCa between 1999 and 2005, who underwent EBRT, BT or radical prostatectomy (RP). Patients with time interval to second malignancy or follow-up shorter than five and two years were excluded for solid and hematopoietic SM analyses respectively. Risks for solid and hematopoietic SM were evaluated via the multivariate Fine and Gray proportional hazards model. Results EBRT and BT resulted in similar increases in solid and hematopoietic SM compared to RP. In subgroup analysis stratified by treatment modality, only the EBRT cohort demonstrated significantly decreased solid and hematopoietic SM in years 2002–2005 compared to years 1999–2001, with adjusted-hazard ratios of 0.752 ( p =0.001) and 0.815 ( p =0.018) respectively. Conclusions EBRT and BT resulted in statistically equivalent increase in both solid and hematopoietic SM compared to RP. EBRT in more recent years resulted in significantly decreased solid and hematopoietic SM, coinciding with increased utilization of IMRT.

Purpose To investigate the accuracy of 4D dose accumulation using projection of dose calculated on the end-exhalation, mid-ventilation, or average intensity breathing phase CT scan, versus dose accumulation performed using full Monte Carlo dose recalculation on every breathing phase. Methods Radiotherapy plans for 10 patients with stage I-II lung cancer were analyzed. All patients had respiratory-correlated computed tomography (4D-CT) performed as part of an IRB-approved research protocol. Stereotactic body radiotherapy (SBRT) plans were optimized using the dose calculated by a commercially-available Monte Carlo algorithm on the end-exhalation 4D-CT phase. 4D dose accumulations using deformable registration were performed with a commercially available tool that projected the planned dose onto every breathing phase without recalculation, as well as with a Monte Carlo recalculation of the dose on all breathing phases. The 3D planned dose (3D-EX), the 3D dose calculated on the average intensity image (3D-AVE), and the 4D accumulations of the dose calculated on the end-exhalation phase CT (4D-PR-EX), the mid-ventilation phase CT (4D-PR-MID), and the average intensity image (4D-PR-AVE), respectively, were compared against the accumulation of the Monte Carlo dose recalculated on every phase. Plan evaluation metrics relating to target volumes and critical structures relevant for lung SBRT were analyzed. Results Plan evaluation metrics tabulated using 4D-PR-EX, 4D-PR-MID, and 4D-PR-AVE differed from those tabulated using Monte Carlo recalculation on every phase by an average of 0.14±0.70 Gy, -0.11±0.51 Gy, and 0.00±0.62 Gy, respectively. Plan evaluation metrics calculated from 3D-EX and 3D-AVE were acceptably accurate for target volumes and most critical structures, however deviations of between 8 and 13 Gy were observed for the proximal bronchial trees of 3 patients. Conclusions The accuracy of 4D dose accumulated by projecting the dose calculated on the end-exhale, mid-ventilation, and average intensity images has been presented for 10 lung cancer SRBT plans. These methods involving projection without re-calculation may be sufficiently accurate compared to 4D dose accumulated from Monte Carlo recalculation on every phase, depending on institutional protocols. Projection of the dose calculated on the mid-ventilation scan was found to be statistically significantly more accurate than projection of the dose calculated on end-exhalation when considering target volume dose metrics. Use of 4D dose accumulation should be considered when evaluating normal tissue complication probabilities as well as in clinical situations where target volumes are directly inferior to mobile critical structures. This article is protected by copyright. All rights reserved.

Summary Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.

Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

Purpose Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells. Methods and Materials Treg cells were identified as a CD4 + CD25 hi Foxp3 + lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT. Results CD4 + CD25 hi Foxp3 + Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression. Conclusions We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

Radiation therapy affects the immune system. In addition to killing radiosensitive immune cells, it can induce functional changes in those cells that survive. Our recent studies showed that the exposure of dendritic cells (DCs) to radiation in vitro influences their ability to present tumour antigen in vivo. Here we show that local radiation therapy of B16 melanoma tumours inhibits the development of systemic immunity to the melanoma antigen MART-1. This inhibition could not be overcome by intratumoral injection of DCs expressing human MART-1 after radiation therapy, suggesting that a form of immune suppression might have developed. On the other hand, injection of MART-expressing DCs prior to tumour irradiation was able to prevent inhibition from developing. These results suggest that local radiation therapy may block the generation of immunity under some circumstances and that strategies may be required to prevent this and allow radiation-induced cell death to translate fully into the development of systemic immunity.

The AD-4/ACE collaboration studies the biological effects of antiprotons with respect to a possible use of antiprotons in cancer therapy. In vitro experiments performed by the collaboration have shown an enhanced biological effectiveness for antiprotons relative to protons. One concern is the normal tissue dose resulting from secondary neutrons produced in the annihilation of antiprotons on the nucleons of the target atoms. Here we present the first organ specific Monte Carlo calculations of normal tissue equivalent neutron dose in antiproton therapy through the use of a segmented CT-based human phantom. The MCNPX Monte Carlo code was employed to quantify the peripheral dose for a cylindrical spread out Bragg peak representing a treatment volume of 1 cm diameter and 1 cm length in the frontal lobe of a segmented whole-body phantom of a 38 year old male. The secondary neutron organ dose was tallied as a function of energy and organ.

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.

© 2015 by Radiation Research Society. Although there has been extensive research done on the biological response to doses of ionizing radiation relevant to radiodiagnostic procedures, very few studies have examined radiation schemes similar to those frequently utilized in CT exams. Instead of a single exposure, CT exams are often made up of a series of scans separated on the order of minutes. DNA damage dose-response kinetics after radiation doses and schemes similar to CT protocols were established in both cultured (ESW-WT3) and whole blood lymphocytes and compared to higher dose exposures. Both the kinetics and extent of H2AX phosphorylation were found to be dose dependent. Damage induction and detection showed a clear dose response, albeit different, at all time points and differences in the DNA repair kinetics of ESW-WT3 and whole blood lymphocytes were characterized. Moreover, using a modified split-dose in vitro experiment, we show that phosphorylation of H2AX is significantly reduced after exposure to CT doses fractionated over a few minutes compared to the same total dose delivered as a single exposure. Because the split-dose exposures investigated here are more similar to those experienced during a CT examination, it is essential to understand why and how these differences occur. This work provides compelling evidence supporting differential biological responses not only between high and low doses, but also between single and multiple exposures to low doses of ionizing radiation.

Resistance of glioblastoma multiforme to radiotherapy poses a major clinical challenge. Farnesyltransferase inhibitors (FTI), such as R115777, have potential to increase radiotherapeutic benefit in this disease, although their mechanism of action is unclear. In our study with eight glioblastoma multiforme cell lines, the most sensitive ones underwent cell cycle arrest in response to FTI treatment. Radiosensitization by FTIs, however, seemed to involve other pathways. If R115777 treatment was initiated

Epidemiological studies suggest that UV exposure from sunlight is the major etiology for skin cancers, both melanocytic and non-melanocytic. However, the radiation-related risk for skin cancer among atomic bomb survivors of Hiroshima and Nagasaki is primarily derived from the excess risk of basal cell carcinoma (BCC), with no demonstrable excess in squamous cell carcinoma or melanoma. The BCCs in this cohort are therefore unusual in being potentially attributable to two types of radiation-UV and ionizing (IR). BCCs have been associated with PTCH and/or p53 tumor suppressor gene alterations. To investigate the roles of these genes in relation to IR and UV exposures, we analyzed both genes in BCC samples from atomic bomb survivors. We examined 47 tumors, of which 70% had non-silent base-substitution p53 mutations independent of IR or UV exposure. However, the distribution of mutation type depends on UV and/or IR exposure. For example, C-to-T transitions at CpG sites adjacent to pyrimidine-pyrimidine (PyPy) sequences were more prevalent in tumors from UV-exposed than UV-shielded body areas and CpG-mutations at non-PyPy sequences were more prevalent in tumors from UV-shielded body areas with high-IR (or=1 Gy) than low-IR (0.2 Gy) exposure. And notably, although p53 deletion-frequencies demonstrated no IR-dose associations, deletions at the PTCH locus were more frequent (79% versus 44%) in tumors with high-IR than low-IR exposure. Moreover, 60% of high-IR tumors harbored both p53 and PTCH abnormalities compared with 23% of low-IR tumors. Therefore, alteration of both genes is likely to play a role in radiation-induced basal cell carcinogenesis.

Radioactive point sources are regularly used for irradiating cell culture and other biological materials. Eccentric rotation is often used to minimize dose disparities that arise from irradiating samples that span a distance from the point source. Rotation provides a great improvement in dose homogeneity compared to inert irradiation yet still presents an obvious shortcoming for exposures in which the sample completes only partial rotation or fractional rotation. In such cases, certain areas of the sample have a closer average distance to the radiation source than other areas within the same sample. This obstacle can be partially overcome by adjusting rotation speed so the sample traverses a full rotation (or multiple thereof) throughout the total irradiation time. Here we investigate the effects of irradiation with eccentric rotation on dose homogeneity. We show that due to the inverse square law that governs dose, even exposures with full rotation result in inhomogeneous dose distributions. This dose inhomogeneity can be substantial, especially for large samples and small source- sample distances. We observed a 33% difference in survival across 100-mm dishes and a 400% difference for 150-mm dishes. The dose inhomogeneity inherent to eccentric rotation increases the actual average dose delivered across the sample compared to that delivered at sample center. We offer a table of correction factors that account for this dose increase and correct the dose delivered at center to the actual average dose delivered across the entire sample.

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-127–35 that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-127–35 also protected mice against growth of B16-A2/Kb tumors in HLA-A2.1/Kb transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.

McBride, W. H., Chiang, C-S., Olson, J. L., Wang, C-C., Hong, J-H., Pajonk, F., Dougherty, G. J., Iwamoto, K. S., Pervan, M. and Liao, Y-P. A Sense of Danger from Radiation. Radiat. Res. 162, 1–19 (2004). Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic “danger” signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of “danger” signals in tissue responses to this agent. This review deals with the nature of putative “danger” signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of “danger” signaling in response to radiation exposure. “Danger” signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alte...

We describe an experiment designed to determine whether or not the densely ionizing particles emanating from the annihilation of antiprotons produce an increase in biological dose in the vicinity of the narrow Bragg peak for antiprotons compared to protons. This experiment is the first direct measurement of the biological effects of antiproton annihilation. The experiment has been approved by the CERN Research Board for running at the CERN Antiproton Decelerator (AD) as AD-4/ACE (Antiproton Cell Experiment) and has begun data taking in June of 2003. The background, description and the current status of the experiment are given. (C) 2004 Elsevier B.V. All rights reserved. 12th International Workshop on Positron and Positronium Physics (POSITRON03), Jul 19-21, 2003, Aarhus Univ, Inst Phys and Astron, Sandbjerg, Denmark

In the last few years, the ubiquitin(Ub)/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded, damaged, and effete proteins. This review examines the role of the Ub/proteasome system in processes underlying the classical effects of irradiation on cells, such as radiation-induced gene expression, DNA repair and chromosome instability, oxidative damage, cell cycle arrest, and cell death. Furthermore, recent evidence suggests that the proteasome is a redox-sensitive target for ionizing radiation and other oxidative stress signals. In other words, the Ub/proteasome system may not simply be a passive player in radiation-induced responses, but may modulate them. The extent of the modulation will be influenced by the functional and structural diversity that is expressed by the system. Cell types vary in the Ub/proteasome structures they possess and the level at which they function, and this changes as they go from the normal to the cancerous condition. Cancer-related functional changes within the Ub/proteasome system may therefore present unique targets for cancer therapy, especially when targeting agents are used in combination with radio- or chemotherapy. The peptide boronic acid compound PS-341, which was designed to inhibit proteasome chymotryptic activity, is in clinical trials for the treatment of solid and hematogenous tumors. It has shown some efficacy on its own and in combination with chemotherapy. Preclinical studies have shown that PS-341 will also potentiate the cytotoxic effects of radiation therapy. In addition, other drugs in common clinical use have been shown to affect proteasome function, and their activities may be valuably reconsidered from this perspective.

Results of this study indicate a radioprotective effect of peroxiredoxin-I. Peroxiredoxin-I is an antioxidant that scavenges hydroperoxides, whereas reactive oxygen species are the main mediators of ionizing radiation toxicity. We hypothesized that peroxiredoxin-I might be induced by cellular exposure to radiation and act to protect them against its cytotoxic effects. Western blot and Northern blot analyses were used to assess peroxiredoxin-I protein and mRNA expression. Rat C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin-I using retroviral vectors. Clonogenic cell survival was used to assess radiosensitivities of the engineered cells. Ionizing radiation induced peroxiredoxin-I protein and mRNA expression in human HT29 colon cancer and rat C6 glioma cells in a dose- and time-dependent manner over a 24 hr period. To determine the effect of peroxiredoxin-I on radiation responses, C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin-I. In clonogenic assays, cells overexpressing peroxiredoxin-I were more radioresistant. Cells transduced with antisense peroxiredoxin-I were marginally more sensitive to radiation toxicity. Irradiation can induce peroxiredoxin-I expression, and the increased peroxiredoxin-I may protect cells from further radiation damage. These results suggest that protection by peroxiredoxin-I may play an important role in the survival of glioma and colon cancer cells in patients undergoing radiation therapy.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RET/PTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RET/PTC1, -2 and -3 are known to be the three major forms. High frequencies of RET/PTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RET/PTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RET/PTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RET/PTC3 was detected only 7 days after X-irradiation, and no transcript of RET/PTC2 was detected. These results are supported by the results of an in vitro study. The RET/PTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RET/PTC3 was induced at a much lower frequency, and no induction of RET/PTC2 was observed. These results suggest that the preferential induction of the RET/PTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

The mechanism and effects of epigenetic alterations in human carcinogenesis are not well understood, except that cancers often have alterations in the methylation status of their genomes. Additionally, human cancers, including aggressive T-cell leukemias and lymphomas, have a high frequency of p53 mutations, particularly missense mutations, which raises the possibility of gain-of-new-function proteins, but the new proteins' oncogenic functions are mechanistically ill-defined. To investigate the mechanisms behind the high prevalence of p53 tumor suppressor gene mutations in aggressive or relapsed T-cell leukemias, we transfected Jurkat cells null for p53 protein with a temperature-sensitive p53 mutant. We showed that this mutant p53 abrogated expression of the T-cell antigen receptor (TCR) by affecting the methylation of an at least 20-kb region of DNA, 5'to the TCR beta-chain gene enhancer region, which includes TCRbetaC1 and betaC2. Expression of the TCR is restored when the temperature is reduced to 32 degrees C, at which temperature the mutant p53 regains wild-type function. The TCR, a common site of dysfunction in T-cell malignancies, is the principal signal transduction moiety controlling both T-cell activation and activation-induced apoptosis. These results suggest a new role for mutant p53-as an epigenetic mutator, bridging p53, methylation, and transcriptional silencing-and suggest novel mechanisms in immunosuppression and cancer progression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

Ionizing radiation is a well-known risk factor of cancer development, but the mechanism of radiation induced carcinogenesis is not clear. Chromosomal rearrangements induced by radiation most likely are one of the principal genetic alterations resulting in malignant transformation. The chimeric BCR-ABL associated with chronic myelogenous leukemia (CML) and H4-RET oncogenes associated with thyroid papillary carcinoma are the result of a translocation and inversion, respectively. In vitro studies showed these genes were induced by high-doses of X-irradiation in cell lines. Studies also show that therapeutic external X-ray doses as high as 60 Gy for treatment of various childhood cancers including Hodgkin's disease significantly increase the risk of thyroid cancer. Therefore, we examined the induction and persistence of these chimeric genes in human thyroid tissues transplanted in scid mice after 50 Gy exposure as a function of time for 2 months to elucidate the early events of thyroid carcinogenesis. The H4-RET genes were detected on day 2 and throughout the 2 month period. On the other hand, BCR-ABL genes were detected on day 2 and were undetectable subsequently. These results suggest that ionizing radiation causes various oncogene activations, but cells with only specific gene alteration uniquely associated with thyroid carcinogenesis are selectively retained demonstrating one of the early events in the beginnings of radiation carcinogenesis in human thyroid tissues.

Brain masses diagnosed in 47 pet dogs as tumors by CT scans, and confirmed in 12 dogs by necropsies, were injected with iodinated contrast media and treated by a modified CT scanner, the CTRx. Twenty-six dogs that received six or more weekly treatments of about 5.6 Gy per fraction, of which about 25% was contributed by radiation from the iodine, for a median total dose of 39 Gy, had a median survival of 230 days. This compares well with the 150 days reported for 25 dogs given 46-48 Gy of cobalt-60 radiation to the whole brain, and is significantly greater than the 6 to 13 days in untreated historic controls.

Four-dimensional (4D) dose calculations for lung cancer radiotherapy have been technically feasible for a number of years but have not become standard clinical practice. The purpose of this study was to determine if clinically significant differences in tumor control probability (TCP) exist between 3D and 4D dose calculations so as to inform the decision whether 4D dose calculations should be used routinely for treatment planning. Radiotherapy plans for Stage I-II lung cancer were created for 8 patients. Clinically acceptable treatment plans were created with dose calculated on the end-exhale 4D computed tomography (CT) phase using a Monte Carlo algorithm. Dose was then projected onto the remaining 9 phases of 4D-CT using the Monte Carlo algorithm and accumulated onto the end-exhale phase using commercially available deformable registration software. The resulting dose-volume histograms (DVH) of the gross tumor volume (GTV), planning tumor volume (PTV), and PTVsetup were compared according to target coverage and dose. The PTVsetup was defined as a volume including the GTV and a margin for setup uncertainties but not for respiratory motion. TCPs resulting from these DVHs were estimated using a wide range of alphas, betas, and tumor cell densities. Differences of up to 5Gy were observed between 3D and 4D calculations for a PTV with highly irregular shape. When the TCP was calculated using the resulting DVHs for fractionation schedules typically used in stereotactic body radiation therapy (SBRT), the TCP differed at most by 5% between 4D and 3D cases, and in most cases, it was by less than 1%. We conclude that 4D dose calculations are not necessary for most cases treated with SBRT, but they might be valuable for irregularly shaped target volumes. If 4D calculations are used, 4D DVHs should be evaluated on volumes that include margin for setup uncertainty but not respiratory motion.

It is widely believed that the anticancer drug 5- fluorouracil (5-FU) must be administered chronically and in low doses to maximize radiosensitization during chemoradiotherapy. The rationale is based upon cell experiments that assumed identical mechanisms of 5-FU action between low-dose chronic (LDC) and high-dose pulsed (HDP) exposures. Here we challenge the paradigm and demonstrate the effectiveness of HDP 5-FU as a radiosensitizer and the wide range of dose/time schedules that can be used to synergize with radiation as compared to the relatively restrictive protocols prescribed for current LDC administrations.Clonogenic survival of human glioblastoma and colon cancer cell lines, U87MG-VIII and HCT-116, respectively, was used to assess temporal and dose effects of 5-FU on radiosensitivity and in split-dose experiments to characterize changes in sublethal damage repair.We show that HDP 5-FU administration does indeed radiosensitize both the highly radioresistant U87MG-VIII and HCT-116. Additionally, we show that this radiosensitization lasts for at least 24 h if cells are pre-irradiated with 2 Gy immediately after HDP 5-FU exposure as a result of a decrease in sublethal damage repair capacity for subsequent irradiations, suggesting the ideal combination of 5-FU bolus injection with fractionation radiotherapy schemes.5-FU bolus administration protocols combined with radiation would not only help improve treatment outcomes and reduce development of 5-FU resistance, but it would greatly benefit patients by shortening clinical stays and lowering overall therapeutic costs.

Using either the colony formation assay or flow cytometry, it is feasible to measure the frequency of rare mutant lymphocytes or erythrocytes in human peripheral blood. Accordingly, we have investigated the mutant cell frequencies of the hypoxanthine-guanine phosphoribosyltransferase and T-cell receptor genes in T lymphocytes and of the glycophorin A gene in erythrocytes of several hundred persons aged 0-96 years. The mutant frequency of every one of these genes increased significantly with age. A simple accumulation of mutations in hematopoietic stem cells over time may explain the age-dependent increase in the frequency of glycophorin A mutants. In contrast, a balance between mutant cell generation and loss should be taken into account for the mechanism of the increase of T-cell mutations.

In the present study, we engrafted normal colonic epithelia and histologically diagnosed colonic adenomas from a familial adenomatous polyposis (FAP) patient into severe combined immunodeficient (SCID) mice and subsequently examined them histologically and molecular biologically. Successful engraftment and metastasis was observed. The facts that human normal colonic epithelium and adenomatous polyps can take in SCID mice indicates the possibility that this human SCID mouse system will be useful for investigating the dynamics of human carcinogenesis in various tissues.

Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks following functional integration into the neuronal network (young GCs), distinguishing them from less active older adult-born GCs and the major population of dentate GCs generated developmentally (together, old GCs). We created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts that was reduced following removal of young GCs by X-ray irradiation. Furthermore, mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation of similar contexts requires adult-born young GCs while old GCs are unnecessary, whereas older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion.

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.

Dose estimates for the assessment of future risks, following accidental exposure to radiation, for certain diseases such as cancer usually rely on both physical and biological quantitative analyses. A traditional biological method of choice is the measurement of chromosome aberration frequencies in peripheral-blood lymphocytes. However, thorough examination of large sample populations is time and labor intensive. Recently, it became possible to measure mutant frequencies in T lymphocytes; one method is a colony assay at the HPRT gene, and the other is a flow-cytometric assay at the T-cell-receptor (TCR) gene. To test for the possible use of these mutation assays, concurrent measurements were taken on blood samples from women who previously received a full course of radiation therapy for gynecological cancer. The results showed that the frequency of TCR mutants correlated reasonably well with that of dicentric chromosomes, whereas the frequency of HPRT mutants did not. Possible uses of the TCR mutation assay in combination with the conventional chromosome analysis or micronucleus assay after exposure of a relatively large population are discussed.

The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q11, resulting in the expression of chimeric BCR-ABL mRNAs specific to chronic myelogenous leukemia (CML). Presence of the fusion gene can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR-ABL fusion genes induced among HL60 cells after 100 Gy of X-irradiation in vitro. In total, five fusion gene transcripts were obtained among 10(8) cells examined. These fusion genes contained not only CML-specific BCR-ABL rearrangements, but also other forms of BCR-ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be direct evidence for the induction of the BCR-ABL fusion gene by X-irradiation. In terms of leukemogenesis, it appears that only those cells bearing certain CML-related BCR-ABL fusion genes are positively selected by virtue of a growth advantage in vivo.

Purpose: To identify exam and patient parameters affecting the biological response to CT studies using in vivo and ex vivo blood samples. Methods: Blood samples were collected under IRB approval from 16 patients undergoing clinically-indicated CT exams. Blood was procured prior to, immediately after and 30minutes following irradiation. A sample of preexam blood was placed on the patient within the exam region for ex vivo analysis. Whole blood samples were fixed immediately following collection and stained for γH2AX to assess DNA damage response (DDR). Median fluorescence of treated samples was compared to non-irradiated control samples for each patient. Patients were characterized by observed biological kinetic response: (a) fast — phosphorylation increased by 2minutes and fell by 30minutes, (b) slow — phosphorylation continued to increase to 30minutes and (c) none — little change was observed or irradiated samples fell below controls. Total dose values were normalized to exam time for an averaged dose-rate in dose/sec for each exam. Relationships between patient biological responses and patient and exam parameters were investigated. Results: A clearer dose response at 30minutes is observed for young patients ( 0.5) compared to old patients (>61yoa; R{sup 2}

Purpose: Discovery of agents that protect or mitigate normal tissue from radiation injury during radiotherapy, accidents, or terrorist attacks is of importance. Specifically, bone marrow insufficiency, with possible infection due to immunosuppression, can occur after total body irradiation (TBI) or regional irradiation and is a major component of the acute radiation syndrome. The purpose of this study was to identify novel radioprotectors and mitigators of the hematopoietic system.Experimental Design: High-throughput screening of small-molecule libraries was done using viability of a murine lymphocyte line as a readout with further validation in human lymphoblastoid cells. The selected compounds were then tested for their ability to counter TBI lethality in mice.Results: All of two major classes of antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were radioprotective. Furthermore, tetracycline protected murine hematopoietic stem/progenitor cell populations from radiation damage and allowed 87.5% of mice to survive when given before and 35% when given 24 h after lethal TBI. Interestingly, tetracycline did not alter the radiosensitivity of Lewis lung cancer cells. Tetracycline and ciprofloxacine also protected human lymphoblastoid cells, reducing radiation-induced DNA double-strand breaks by 33% and 21%, respectively. The effects of these agents on radiation lethality are not due to the classic mechanism of free radical scavenging but potentially through activation of the Tip60 histone acetyltransferase and altered chromatin structure.Conclusions: Tetracyclines and fluoroquinolones can be robust radioprotectors and mitigators of the hematopoietic system with potential utility in anticancer radiotherapy and radiation emergencies. (Clin Cancer Res 2009;15(23):7238–45)