William DeGraff

Ionizing Radiation-Induced Oxidative Stress Alters miRNA Expression

Background MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H2O2). Methodology/Principal Findings Normal human fibroblasts were exposed to radiation, H2O2, or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H2O2 treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation. Conclusions These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H2O2, and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, Tempol

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.

Chemosensitivity testing of human lung cancer cell lines using the MTT assay.

Thirty human lung cancer cell lines were tested for chemosensitivity using the semi-automated, non-clonogenic MTT assay. The tumour cell lines came from three major categories of patients: untreated small cell lung cancer (SCLC); SCLC relapsing on chemotherapy; and non-SCLC predominantly from untreated patients. From these data IC50 values were derived for each drug in each cell line. While some inter-experimental variability was observed, the rank order of chemosensitivity of each cell line within this panel was significantly correlated between experiments. These results show that tumour cell lines derived from untreated small cell lung cancer patients were the most chemosensitive for adriamycin, melphalan, vincristine and VP16 compared to the other cell types. In addition, untreated SCLC was more sensitive than non-SCLC to BCNU and cis-platin, while vincristine was the only drug to which treated SCLC was more sensitive compared to the non-SCLC lines. In contrast, no significant differences between the lung cancer types were observed for vinblastine. Thus, this panel of lung cancer cells exhibited a drug sensitivity profile paralleling that observed in clinical practice. These results suggest that this lung cancer cell line panel in combination with a relatively simple but reproducible chemosensitivity assay, such as the MTT assay, has potential for the testing of drug combinations and evaluating new anti-cancer agents in vitro.

Gene Expression Profiling of Breast, Prostate, and Glioma Cells following Single versus Fractionated Doses of Radiation

Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 x 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-beta-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures.

Radiation sensitivity of human lung cancer cell lines

X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2 Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens.

Superoxide Fluxes Limit Nitric Oxide-induced Signaling

Independently, superoxide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document}) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1α, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1α, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document}.

Nitroxide Sod-Mimics: Modes of Action

Low molecular weight superoxide dismutase mimics have been shown to afford protection from oxidative damage. Such SOD-mimics can readily permeate cell membrane achieving sufficiently high levels both inside and outside the cell to effectively detoxify intracellular O2-.. Preliminary findings also indicated that metal-based and metal-free SOD-mimics can protect hypoxic cells from H2O2-induced damage. The present study explored the possibility that SOD-mimics such as desferrioxamine-Mn(III) chelate [DF-Mn] or cyclic nitroxide stable free radicals could protect from O2-.-independent damage. Killing of monolayered V79 Chinese hamster cells was induced by H2O2 or by t-butyl hydroperoxide (t-BHP) and assayed clonogenically. Neither catalase nor native SOD protected the cells from t-BHP. In contrast, both DF-Mn and cyclic nitroxides protected suggesting cytotoxic processes independent of O2-. or of O2-.-derived active species. The inhibition of the damage by both metal-free and metal-based SOD mimics is attributable to either SOD-mimic reacting with reduced transition metal to block the Fenton reaction and/or intercepting and detoxifying intracellular organic free radicals.

The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.

Radioprotection in Normal Tissue and Delayed Tumor Growth by Blockade of CD47 Signaling

Inhibition of a membrane receptor protects normal tissue from radiation injury and simultaneously enhances the ability of ionizing radiation to delay tumor growth. One major caveat of radiation therapy in cancer treatment is that the effective dose delivered to the individual is oftentimes necessarily limited to avoid major side effects that arise from collateral damage inflicted on surrounding normal tissue. Efforts to devise methods to sensitize tumor tissue to radiation injury or to protect normal tissue by scavenging for reactive by-products of radiation have only been moderately successful, as their broad clinical utility is hampered by a lack of specificity or by toxicity. Now, a team of researchers describes an approach to protecting normal human cells from high-dose radiation damage while, at the same time, increasing the sensitivity of tumor cells to radiation. We know that the pathway triggered by the secretory glycoprotein thrombospondin-1 (TSP1) and its corresponding membrane receptor CD47 in response to injury limits the survival of vascular cells and tissues. In this work, Roberts et al. have devised a strategy by which the systemic blockade of TSP1 and its receptor CD47 inhibits downstream signaling functions to protect against radiation injury the highly radiation-sensitive endothelial cells that line the lumen of the vasculature. More important, they show in mice injected with human tumors that suppression of CD47 by systemic administration of an antisense CD47 morpholino can sensitize the tumors to high-dose radiation therapy while keeping collateral damage at bay, exemplified in part by the resilience of the skin, muscle, and bone to radiation injury. Their experiments simultaneously show a significant delay in the time that these tumors take to grow back. This phenomenon remains to be explained, but there is an improvement in vascular function in irradiated, CD47-suppressed mice, and, at the cellular level, in vivo stem cells are still viable and proliferate, whereas peripheral immune cells, which infiltrate the inflammatory tumor microenvironment, are protected and recruited to the site. Although it remains speculative how CD47 participates in antitumor immunity within this experimental context, combined treatment of high-dose radiation and CD47 suppression in these translational studies suggests that a more aggressive therapeutic irradiation strategy with concurrent protection of neighboring normal tissue is possible. Testing the effectiveness of such a treatment strategy will be required to see whether this approach is useful. Radiation-induced damage of normal tissues restricts the therapeutic doses of ionizing radiation that can be delivered to tumors and thereby limits the effectiveness of radiotherapy. Thrombospondin-1 signaling through its cell surface receptor CD47 limits recovery from several types of stress, and mice lacking either gene are profoundly resistant to radiation injury. We describe strategies to protect normal tissues from radiation damage with antibodies to CD47 or thrombospondin-1, a CD47-binding peptide, or antisense suppression of CD47. A morpholino oligonucleotide targeting CD47 confers radioresistance to human endothelial cells in vitro and protects soft tissue, bone marrow, and tumor-associated leukocytes in irradiated mice. In contrast, CD47 suppression in mice bearing melanoma or squamous lung tumors before irradiation results in 89% and 71% smaller tumors, respectively. Thus, inhibition of CD47 signaling maintains the viability of normal tissues after irradiation while increasing the radiosensitivity of tumors.

Further evidence for distinct reactive intermediates from nitroxyl and peroxynitrite: effects of buffer composition on the chemistry of Angeli's salt and synthetic peroxynitrite

The nitroxyl (HNO) donor Angelis salt (Na(2)N(2)O(3); AS) is cytotoxic in vitro, inducing double strand DNA breaks and base oxidation, yet may have pharmacological application in the treatment of cardiovascular disease. The chemical profiles of AS and synthetic peroxynitrite (ONOO(-)) in aerobic solution were recently compared, and AS was found to form a distinct reactive intermediate. However, similarities in the chemical behavior of the reactive nitrogen oxide species (RNOS) were apparent under certain conditions. Buffer composition was found to have a significant and unexpected impact on the observed chemistry of RNOS, and varied buffer conditions were utilized to further distinguish the chemical profiles elicited by the RNOS donors AS and synthetic ONOO(-). Addition of HEPES to the assay buffer significantly quenched oxidation of dihydrorhodamine (DHR), hydroxylation of benzoic acid (BA), and DNA damage by both AS and ONOO(-), and oxidation and nitration of hydroxyphenylacetic acid by ONOO(-). Additionally, H(2)O(2) was produced in a concentration-dependent manner from the interaction of HEPES with both the donor intermediates. Interestingly, clonogenic survival was not affected by HEPES, indicating that H(2)O(2) is not a contributing factor to in vitro cytotoxicity of AS. Variation in RNOS reactivity was dramatic with significantly higher relative affinity for the AS intermediate toward DHR, BA, DNA, and HEPES and increased production of H(2)O(2). Further, AS reacted to a significantly greater extent with the unprotonated amine form of HEPES while the interaction of ONOO(-) with HEPES was pH-independent. Addition of bicarbonate only altered ONOO(-) chemistry. This study emphasizes the importance of buffer composition on chemical outcome and thus on interpretation and provides further evidence that ONOO(-) is not an intermediate formed between the reaction of O(2) and HNO produced by AS.