Ross B. Mikkelsen

Inhibition of Protein-tyrosine Phosphatases by Mild Oxidative Stresses Is Dependent on S-Nitrosylation

Previous studies have shown that a Ca2+-dependent nitric-oxide synthase (NOS) is activated as part of a cellular response to low doses of ionizing radiation. Genetic and pharmacological inhibitor studies linked this NO signaling to the radiation-induced activation of ERK1/2. Herein, a mechanism for the radiation-induced activation of Tyr phosphorylation-dependent pathways (e.g. ERK1/2) involving the inhibition of protein-Tyr phosphatases (PTPs) by S-nitrosylation is tested. The basis for this mechanism resides in the redox-sensitive active site Cys in PTPs. These studies also examined oxidative stress induced by low concentrations of H2O2. S-Nitrosylation of total cellular PTP and immunopurified SHP-1 and SHP-2 was detected as protection of PTP enzymatic activity from alkylation by N-ethylmaleimide and reversal by ascorbate. Both radiation and H2O2 protected PTP activity from alkylation by a mechanism reversible by ascorbate and inhibited by NOS inhibitors or expression of a dominant negative mutant of NOS-1. Radiation and H2O2 stimulated a transient increase in cytoplasmic free [Ca2+]. Radiation, H2O2, and the Ca2+ ionophore, ionomycin, also stimulated NOS activity, and this was associated with an enhanced S-nitrosylation of the active site Cys453 determined by isolation of S-nitrosylated wild type but not active site Cys453 → Ser SHP-1 mutant by the “biotin-switch” method. Thus, one consequence of oxidative stimulation of NO generation is S-nitrosylation and inhibition of PTPs critical in cellular signal transduction pathways. These results support the conclusion that a mild oxidative signal is converted to a nitrosative one due to the better redox signaling properties of NO.

Protein tyrosine nitration in cellular signal transduction pathways

How specificity and reversibility in tyrosine nitration are defined biologically in cellular systems is poorly understood. As more investigations identify proteins involved in cell regulatory pathways in which only a small fraction of that protein pool is modified by nitration to affect cell function, the mechanisms of biological specificity and reversal should come into focus. In this review experimental evidence has been summarized to suggest that tyrosine nitration is a highly selective modification and under certain physiological conditions fulfills the criteria of a physiologically relevant signal. It can be specific, reversible, occurs on a physiological time scale, and, depending on a target, can result in either activation or inhibition.

Factors influencing protein tyrosine nitration--structure-based predictive models.

Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high-resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged side chain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines for which there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases, predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives).

Nitric oxide synthase inhibition enhances the antitumor effect of radiation in the treatment of squamous carcinoma xenografts.

This study tests whether the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA), combines favorably with ionizing radiation (IR) in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01). Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control.

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression

Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased β-catenin expression, and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-d-glucose–deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth–promoting and antiapoptotic signaling properties. Implications: The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth. Mol Cancer Res; 13(6); 1034–43. ©2015 AACR.

Proteomic Analysis of Radiation-Induced Changes in Rat Lung: Modulation by the Superoxide Dismutase Mimetic MnTE-2-PyP5+

PURPOSEnTo identify temporal changes in protein expression in the irradiated rat lung and generate putative mechanisms underlying the radioprotective effect of the manganese superoxide dismutase mimetic MnTE-2-PyP(5+).nnnMETHODS AND MATERIALSnFemale Fischer 344 rats were irradiated to the right hemithorax with a single dose of 28 Gy and killed from day 1 to 20 weeks after irradiation. Proteomic profiling was performed to identify proteins that underwent significant changes in abundance. Some irradiated rats were administered MnTE-2-PyP(5+) and changes in protein expression and phosphorylation determined at 6 weeks after irradiation.nnnRESULTSnRadiation induced a biphasic stress response in the lung, as shown by the induction of heme oxygenase 1 at 1-3 days and at 6-8 weeks after irradiation. At 6-8 weeks after irradiation, the down-regulation of proteins involved in cytoskeletal architecture (filamin A and talin), antioxidant defense (biliverdin reductase and peroxiredoxin II), and cell signaling (β-catenin, annexin II, and Rho-guanosine diphosphate dissociation inhibitor) was observed. Treatment with MnTE-2-PyP(5+) partially prevented the apparent degradation of filamin and talin, reduced the level of cleaved caspases 3 and 9, and promoted Akt phosphorylation as well as β-catenin expression.nnnCONCLUSIONnA significant down-regulation of proteins and an increase in protein markers of apoptosis were observed at the onset of lung injury in the irradiated rat lung. Treatment with MnTE-2-PyP(5+), which has been demonstrated to reduce lung injury from radiation, reduced apparent protein degradation and apoptosis indicators, suggesting that preservation of lung structural integrity and prevention of cell loss may underlie the radioprotective effect of this compound.

Role of Interleukin-1 in Radiation-Induced Cardiomyopathy

Thoracic X-ray therapy (XRT), used in cancer treatment, is associated with increased risk of heart failure. XRT-mediated injury to the heart induces an inflammatory response leading to cardiomyopathy. The aim of this study was to determine the role of inter-leukin (IL)-1 in response to XRT injury to the heart and on the cardiomyopathy development in the mouse. Female mice with genetic deletion of the IL-1 receptor type I (IL-1R1 knockout mice [IL-1R1 KO]) and treatment with recombinant human IL-1 receptor antagonist anakinra, 10 mg/kg twice daily for 7 d, were used as independent approaches to determine the role of IL-1. Wild-type (wt) or IL-1R1 KO mice were treated with a single session of XRT (20 or 14 gray [Gy]). Echocardiography (before and after isoproterenol challenge) and left ventricular (LV) catheterization were performed to evaluate changes in LV dimensions and function. Masson’s trichrome was used to assess myocardial fibrosis and pericardial thickening. After 20 Gy, the contractile reserve was impaired in wt mice at d 3, and the LV ejection fraction (EF) was reduced after 4 months when compared with sham-XRT. IL-1R1 KO mice had preserved contractile reserve at 3 d and 4 months and LVEF at 4 months after XRT. Anakinra treatment for 1 d before and 7 d after XRT prevented the impairment in contractile reserve. A significant increase in LV end-diastolic pressure, associated with increased myocardial interstitial fibrosis and pericardial thickening, was observed in wt mice, as well as in IL-1R1 KO-or anakinra-treated mice. In conclusion, induction of IL-1 by XRT mediates the development of some, such as the contractile impairment, but not all aspects of the XRT-induced cardiomyopathy, such as myocardial fibrosis or pericardial thickening.

Current Status and Recommendations for the Future of Research, Teaching, and Testing in the Biological Sciences of Radiation Oncology: Report of the American Society for Radiation Oncology Cancer Biology/Radiation Biology Task Force, Executive Summary

In early 2011, a dialogue was initiated within the Board of Directors (BOD) of the American Society for Radiation Oncology (ASTRO) regarding the future of the basic sciences of the specialty, primarily focused on the current state and potential future direction of basic research within radiation oncology. After consideration of the complexity of the issues involved and the precise nature of the undertaking, in August 2011, the BOD empanelled a Cancer Biology/Radiation Biology Task Force (TF). The TF was charged with developing an accurate snapshot of the current state of basic (preclinical) research in radiation oncology from the perspective of relevance to the modern clinical practice of radiation oncology as well as the education of our trainees and attending physicians in the biological sciences. The TF was further charged with making suggestions as to critical areas of biological basic research investigation that might be most likely to maintain and build further the scientific foundation and vitality of radiation oncology as an independent and vibrant medical specialty. It was not within the scope of service of the TF to consider the quality of ongoing research efforts within the broader radiation oncology space, to presume to consider their future potential, or to discourage in any way the investigators committed to areas of interest other than those targeted. The TF charge specifically precluded consideration of research issues related to technology, physics, or clinical investigations. This document represents an Executive Summary of the Task Force report.

Abstract LB-339: Cause of death in cancer survivors

Background: Cancer, in general, is regarded as one of the most life threatening diseases. In the past decade, with the application of advanced scientific and medical technologies for cancer early detection, prevention, and treatment, cancer survivors are now living much longer and do not died directly from cancer, but from other diseases and complications, therefore, becomes increasingly important to understand major causes of death among cancer survivors to improve the quality of life and prolong life expectancy of cancer survivors. We conducted a study to investigate the cause of death among cancer survivors using data from national surveys. Methods: The analytical population consist of 1807 cancer survivors identified from the National Health and Nutrition Examination Survey (NHANES) III 1988-1994 and NHANES 1999- 2004. We excluded participants with age less than 18 years old and those with skin cancer. Cancer survivors were identified if they self-reported having ever been told by a doctor or other health professional that he/she had cancer or a malignancy of any kind. The cause of death was ascertained through the National Death Index (NDI). All participants were followed from the date of survey to the date of the survey to December 31, 2006 by linking NDI death certificate records. Results: The Follow-up time ranged from 0 to 17.3 years. Total 776 participants died during the follow-up period. Underlying cause of death were recoded as 113 death causes in the National Death Index. We classified all death causes into 3 categories: related with cancer, indeterminate and not related with cancer. The causes of death were stratified by the duration since the diagnosis of cancer, which was calculated as follow-up time plus years between age at NHANES survey and age when being diagnosed with cancer. Commonly reported types of cancer were prostate (n=165), breast (n=141) and colorectal cancer (n=117) among 761 male and 1046 female participants. At time of the survey, there were 17.7% and 26.5% with diabetes, 63.2 and 66.9 with cardiovascular condition, and 58.7 and 62.9 had hypertension, and 61.3% and 70.5% with hypercholesterolemia in men and women, respectively. Among the deseeded cancer survivor, 41.8%, 9.3, and 48.9% were died from cancer, indeterminate, disease not related with cancer. The percentages were 43.4%, 6.2% and 50.4%, respectively in men and 40.4%, 12.1% and 47.6%, respectively in women. Noticeably, with the increase of the duration since diagnosis of cancer, higher percentage of cancer survivor died from disease not related to cancer, ranging from 23.3% within 5 years to 72.1% in more than 30 years (p value for trend Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-339. doi:1538-7445.AM2012-LB-339

Redox signaling mechanisms and radiationinduced bystander effects

In many ways the questions posed for this Commentary are questions of how cells sense and respond to ionization events. I will focus on how cells sense radiation-induced ionization events and how cells amplify the signal emanating from these events and relay these signals to adjacent cells. Knowledge of these signal transduction mechanisms is important for understanding the diverse nature of bystander effects (from cytoprotective to cytotoxic), and the different bystander effects observed depending on whether the nucleus or cytoplasm are irradiated. Sensitive mechanisms have evolved for detecting the consequence of radiation-induced damage. The most studied are nuclear. The response to a single DSB is highly amplified and rapid and initially involves the phosphorylation of hundreds to thousands of histone H2AX molecules surrounding the DSB. This results in the recruitment to the site of a number of proteins involved in DNA repair and in downstream signaling to cell cycle checkpoints and transcriptional responses. At high radiation doses, DSBs are repaired in cells that survive the radiation exposure. However, in cells that are irradiated with low doses of radiation so that there is B/1 DSB per cell and that are maintained in a nonproliferative state, the DSB persists unrepaired for days. This may provide one mechanism of continuous stress signaling to adjacent cells. Less is known about the consequences of cytoplasmic irradiation. Selective cytoplasmic irradiation with an a -particle microbeam induces a mutation spectrum different from nuclear irradiation and similar to the mutations spontaneously produced by endogenous metabolism. Exposure to ionizing radiation also rapidly activates several signal transduction pathways by mechanisms that can best be explained as involving cytoplasmic ionization events. These include growth factor receptors, changes in cytoplasmic Ca levels, and stress-response kinases. ] 11 Ward questioned how cells sense and amplify the few primary ionization events at clinically relevant doses (:/2000/Gy/cell) resulting in the rapid and robust activation of cellular signal transduction pathways. The calculated amounts of primary and secondary reactive oxygen species (ROS) generated by irradiation are insignificant compared witho the amount produced by metabolism. However, actual measurements of ROS/RNS (reactive nitrogen species) post-irradiation indicate much higher ROS/RNS amounts are produced than predicted and suggest possible sensing/amplification mechanisms. ] 17 Fluorescent dyes sensitive to ROS/RNS reveal that high and low LET radiation stimulate ROS/RNS generation within minutes of a radiation exposure in diverse cell types. A single cell analysis by digitized fluorescence microscopy demonstrates that radiation (Sr) stimulated ROS/RNS generation within seconds of starting radiation treatment (1]/10 Gy) that persisted for 2]/5 min post-irradiation. Whereas the amount of ROS/RNS generated per cell remains relatively constant over this radiation dose range, the numbers of responding cells increase with dose. A semi-log plot of responding cells versus dose is a straight line extrapolating to 1, consistent with a single target. Although this does not appear to support a bystander effect it is important to note both the early post-irradiation time points examined and that the cells were subconfluent. What is the target(s)? Experiments using diphenyliodonium (DPI) suggest that a -particle irradiation activates NADPH oxidase stimulating O2 and H2O2 generation. However, DPI, an FAD analog, inhibits several enzymes, including those of mitochondrial complex I. Studies with cells lacking mitochondrial DNA and deficient in electron transport and with inhibitors of mitochondrial permeability transition suggest that mitochondria represent the sensor of radiation-induced ionization events. The me*Correspondence: Ross Mikkelsen, Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA 23298-0058, USA E-mail: swkelly@hsc.vcu.edu Human & Experimental Toxicology (2004) 23: 75 ]/79