Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

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 interleukin (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.

Sepiapterin ameliorates chemically induced murine colitis and azoxymethane-induced colon cancer.

The effects of modulating tetrahydrobiopterin (BH4) levels with a metabolic precursor, sepiapterin (SP), on dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal cancer were studied. SP in the drinking water blocks DSS-induced colitis measured as decreased disease activity index (DAI), morphologic criteria, and recovery of Ca(2+)-induced contractility responses lost as a consequence of DSS treatment. SP reduces inflammatory responses measured as the decreased number of infiltrating inflammatory macrophages and neutrophils and decreased expression of proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and IL-17A. High-performance liquid chromatography analyses of colonic BH4 and its oxidized derivative 7,8-dihydrobiopterin (BH2) are inconclusive although there was a trend for lower BH4:BH2 with DSS treatment that was reversed with SP. Reduction of colonic cGMP levels by DSS was reversed with SP by a mechanism sensitive to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). ODQ abrogates the protective effects of SP on colitis. This plus the finding that SP reduces DSS-enhanced protein Tyr nitration are consistent with DSS-induced uncoupling of NOS. The results agree with previous studies that demonstrated inactivation of sGC in DSS-treated animals as being important in recruitment of inflammatory cells and in altered cholinergic signaling and colon motility. SP also reduces the number of colon tumors in AOM/DSS-treated mice from 7 to 1 per unit colon length. Thus, pharmacologic modulation of BH4 with currently available drugs may provide a mechanism for alleviating some forms of colitis and potentially minimizing the potential for colorectal cancer in patients with colitis.

Radiation induces ESCRT pathway dependent CD44v3+ extracellular vesicle production stimulating pro-tumor fibroblast activity in breast cancer.

Despite recent advances in radiotherapeutic strategies, acquired resistance remains a major obstacle, leading to tumor recurrence for many patients. Once thought to be a strictly cancer cell intrinsic property, it is becoming increasingly clear that treatment-resistance is driven in part by complex interactions between cancer cells and non-transformed cells of the tumor microenvironment. Herein, we report that radiotherapy induces the production of extracellular vesicles by breast cancer cells capable of stimulating tumor-supporting fibroblast activity, facilitating tumor survival and promoting cancer stem-like cell expansion. This pro-tumor activity was associated with fibroblast production of the paracrine signaling factor IL-6 and was dependent on the expression of the heparan sulfate proteoglycan CD44v3 on the vesicle surface. Enzymatic removal or pharmaceutical inhibition of its heparan sulfate side chains disrupted this tumor-fibroblast crosstalk. Additionally, we show that the radiation-induced production of CD44v3+ vesicles is effectively silenced by blocking the ESCRT pathway using a soluble pharmacological inhibitor of MDA-9/Syntenin/SDCBP PDZ1 domain activity, PDZ1i. This population of vesicles was also detected in the sera of human patients undergoing radiotherapy, therefore representing a potential biomarker for radiation therapy and providing an opportunity for clinical intervention to improve treatment outcomes.

Mitigation of Radiation-Induced Lung and Heart Injuries in Mice by Oral Sepiapterin after Irradiation.

After radiation exposure, endothelium-dependent vasorelaxation is impaired due to impaired nitric oxide production. Endothelial dysfunction is characterized by uncoupled endothelial nitric oxide synthase activity, oxidation of the reduced cofactor tetrahydrobiopterin to dihydrobiopterin as one well recognized mechanism. Oral treatment with sepiapterin, a tetrahydrobiopterin precursor, decreased infiltrating inflammatory cells and cytokine levels in mice with colitis. We therefore tested whether a synthetic sepiapterin, PTC923, might mitigate radiation-induced cardiac and pulmonary injuries. C57L/J wild-type 6-8-week-old mice of both sexes received 5 Gy total-body irradiation (TBI), followed by a top-up dose of 6.5 Gy to the thorax (total thoracic dose of 11.5 Gy). Starting from 24 h postirradiation, mice were treated once daily with 1 mg/kg PTC923 for six days by oral gavage. Assessment of lung injury by breathing rate was measured every other week and echocardiography to assess heart function was performed at different time points (8, 30, 60, 90 and 180 days). Plasma proteins (fibrinogen, neutrophil elastase, C-reactive protein, and IL-6) were assessed as well. TBI induced a reduction in cardiac contractile reserve and an impairment in diastolic function restored by daily oral PTC923. Postirradiation lung injury was significantly delayed by PTC923. TBI mice treated with PTC923 experienced a longer survival compared to nonirradiated mice (71% vs. 40% of mice alive after 180 days). PTC923-treated mice showed a reduction in inflammatory mediators, especially IL-6 and IL-1b. In conclusion, these findings support the proposal that PTC923 is a potential mitigator of cardiac and lung injury caused by TBI.

Nitration of the tumor suppressor protein p53 at tyrosine 327 promotes p53 oligomerization and activation.

Previous studies demonstrate that nitric oxide (NO) promotes p53 transcriptional activity by a classical DNA damage responsive mechanism involving activation of ATM/ATR and phosphorylation of p53. These studies intentionally used high doses of NO donors to achieve the maximum DNA damage. However, lower concentrations of NO donors also stimulate rapid and unequivocal nuclear retention of p53 but apparently do not require ATM/ATR-dependent p53 phosphorylation or total p53 protein accumulation. To identify possible mechanisms for p53 activation at low NO levels, the role of Tyr nitration in p53 activation was evaluated. Low concentrations of the NO donor, DETA NONOate (<200 microM), exclusively nitrate Tyr327 within the tetramerization domain promoting p53 oligomerization, nuclear accumulation, and increased DNA-binding activity without p53 Ser15 phosphorylation. Molecular modeling indicates that nitration of one Tyr327 stabilizes the dimer by about 2.67 kcal mol(-1). Significant quantitative and qualitative differences in the patterns of p53-target gene modulation by low (50 microM), non-DNA-damaging and high (500 microM), DNA-damaging NO donor concentrations were shown. These results demonstrate a new posttranslational mechanism for modulating p53 transcriptional activity responsive to low NO concentrations and independent of DNA damage signaling.

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.

A Phase II Study to Prevent Radiation-induced Rectal Injury With Lovastatin.

PURPOSE: Physician reported symptomatic late rectal injury occurs in about 5% to 25% of patients treated with radiation therapy for prostate cancer, depending on the treatment technique. Patients, however, report clinically meaningful declines in bowel/rectal function regardless of the technique used. Lovastatin has been shown to protect mice from late radiation injury. This study was designed to determine if lovastatin might reduce the incidence of late rectal injury in patients receiving radiation therapy for prostate cancer. MATERIALS AND METHODS: Patients with adenocarcinoma of the prostate receiving radiotherapy with curative intent were eligible. A portion of the rectum had to receive at least 60 Gy. Gastrointestinal functioning was assessed using both physician-reported and patient-reported instruments at baseline and at prescribed intervals during and after treatment. Lovastatin (20 to 80 mg/d) was started on day 1 of radiation and continued for 12 months. Patients were followed for an additional 12 months. The primary endpoint was physician-reported rectal toxicity ≥grade 2 during the first 2 years after treatment. RESULTS: A total of 20/53 (38%) patients developed grade 2 or higher toxicity during the 2-year follow-up period. Seventeen patients had 1 or more unresolved gastrointestinal symptom at the end of 2 years, 3 (6%) of which were grade 2 and none were of higher grade. CONCLUSIONS: The primary endpoint of the study was not met. Lovastatin, as administered in this trial, did not reduce the incidence of grade 2 or higher rectal toxicity compared with historical controls.

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms.

In the past few years, nuclear DNA damage-sensing mechanisms activated by ionizing radiation have been identified, including ATM/ATR and the DNA-dependent protein kinase. Less is known about sensing mechanisms for cytoplasmic ionization events and how these events influence nuclear processes. Several studies have demonstrated the importance of cytoplasmic signaling pathways in cytoprotection and mutagenesis. For cytoplasmic signaling, radiation-stimulated reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential activators of these pathways. This review summarizes recent studies on the chemistry of radiation-induced ROS/RNS generation and emphasizes interactions between ROS and RNS and the relative roles of cellular ROS/RNS generators as amplifiers of the initial ionization events. Cellular mechanisms for regulating ROS/RNS levels are discussed. The mechanisms by which cells sense ROS/RNS are examined in terms of how ROS/RNS modify protein structure and function, for example, interactions with metal-thiol clusters, protein tyrosine nitration, protein cysteine oxidation, S-thiolation and S-nitrosylation. We propose that radiation-induced ROS are the initiators and that nitric oxide (NO*) or derivatives are the effectors activating these signal transduction pathways. In responding to cellular ionization events, the cell converts an oxidative signal to a nitrosative one because ROS are too reactive and unspecific in their reactions for regulatory purposes and the cell is equipped to precisely modulate NO* levels.

Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells.

In this study we have investigated the effects of low dose ionizing radiation (2 Gy) on p70 S6 kinase and Akt signaling with respect to Erb-B receptors in both the A431 squamous and the MDA-MB-231 mammary carcinoma cell lines. Ionizing radiation caused a 2-3-fold increase in p70 S6 kinase activity that was blocked pharmacologically using an EGFR inhibitor (AG1478) alone, or in combination with an Erb-B2 inhibitor (AG825). These results suggested that both EGFR and Erb-B2 receptors could initiate radiation-induced activation of p70 S6K. EGFR dependent Erb-B3 signaling also contributed to p70 S6 kinase activity through recruitment and activation of PI3K, which has been shown to regulate p70 S6 kinase activity. Furthermore, inhibition of the EGFR blocked IR stimulated increases in protein translation, a biologic consequence of p70 S6 kinase activation. We also report that ionizing radiation stimulated Akt activity that was partially independent of PI3K activity, but dependent on Erb-B2 function. Erb-B2 inhibition also correlated with enhanced apoptosis following IR exposure, suggesting an important role for Erb-B2 in cell survival. Together this work demonstrates that the Erb-B receptor tyrosine kinase network stimulates cytoprotective p70 S6 kinase and Akt activity in response to clinically relevant doses of ionizing radiation.

Inhibition of the type III epidermal growth factor receptor variant mutant receptor by dominant-negative EGFR-CD533 enhances malignant glioma cell radiosensitivity.

PURPOSE: The commonly expressed variant epidermal growth factor receptor (EGFR), the type III EGFR variant (EGFRvIII), functions as an oncoprotein promoting neoplastic transformation and tumorigenicity. The role of EGFRvIII in cellular responses to genotoxic stress, such as ionizing radiation, is only minimally defined. Thus, we have investigated EGFRvIII as a potential modulator of cellular radiation responses and explored the feasibility of adenovirus (Ad)-mediated expression of dominant-negative EGFR-CD533 as a gene therapeutic approach for inhibiting EGFRvIII function in vitro and in vivo. EXPERIMENTAL DESIGN AND RESULTS: EGFR-CD533 and EGFRvIII were expressed in vitro and in vivo in malignant U-373 MG glioma cells through transduction with an Ad vector, Ad-EGFR-CD533 and Ad-EGFRvIII, respectively. In vivo studies defined the importance of EGFRvIII as a modulator of radiation responses, demonstrating a 2.6-fold activation of EGFRvIII in U-373 malignant glioma tumors. Concomitant expression of EGFR-CD533 inhibited the radiation-induced activation of EGFRvIII in vitro and completely abolished the enhanced clonogenic survival conferred by EGFRvIII. The ability of EGFR-CD533 to inhibit EGFRvIII function was further confirmed in vivo through complete inhibition of EGFRvIII-mediated increased tumorigenicity and radiation-induced activation of EGFRvIII. Growth delay assays with U-373 xenograft tumors demonstrated that the expression of EGFR-CD533 significantly enhanced radiosensitivity of tumor cells under conditions of intrinsic and Ad-mediated EGFRvIII expression. CONCLUSIONS: We conclude that EGFRvIII confers significant radioresistance to tumor cells through enhanced cytoprotective responses, and we have demonstrated that dominant-negative EGFR-CD533 effectively inhibits EGFRvIII function. These data affirm the broad potential of EGFR-CD533 to radiosensitize human malignant glioma cells.

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression.

UNLABELLED: 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 ß-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.

A Preliminary Study on Racial Differences in HMOX1, NFE2L2, and TGFß1 Gene Polymorphisms and Radiation-Induced Late Normal Tissue Toxicity.

PURPOSE: This study tested whether racial differences in genetic polymorphisms of 4 genes involved in wound repair and response to radiation can be used to predict the occurrence of normal tissue late effects of radiation therapy and indicate potential therapeutic targets. METHODS AND MATERIALS: This prospective study examined genetic polymorphisms that modulate the expression of 4 genes involved in inflammation and fibrosis and response to radiation (HMOX1, NFE2L2, NOS3, and TGFß1). DNA from blood samples of 179 patients (∼ 80% breast and head and neck) collected at the time of diagnosis by their radiation oncologist as exhibiting late normal tissue toxicity was used for the analysis. Patient demographics were as follows: 56% white, 43% African American, 1% other. Allelic frequencies of the different polymorphisms of the participants were compared with those of the general American population stratified by race. Twenty-six additional patients treated with radiation, but without toxicity at 3 months or later after therapy, were also analyzed. RESULTS: Increased frequency of a long GT repeat in the HMOX1 promoter was associated with late effects in both African American and white populations. The single nucleotide polymorphisms (SNP) rs1800469 in the TGFß1 promoter and the rs6721961 SNP in the NFE2L2 promoter were also found to significantly associate with late effects in African Americans but not whites. A combined analysis of these polymorphisms revealed that >90% of African American patients with late effects had at least 1 of these minor alleles, and 58% had 2 or more. No statistical significance was found relating the studied NOS3 polymorphisms and normal tissue toxicity. CONCLUSIONS: These results support a strong association between wound repair and late toxicities of radiation. The presence of these genetic risk factors can vary significantly among different ethnic groups, as demonstrated for some of the SNPs. Future studies should account for the possibility of such ethnic heterogeneity in the late toxicities of radiation.

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.

Physical activity and sedentary behavior of cancer survivors and non-cancer individuals: results from a national survey.

Increasing physical activity and decreasing sedentary behavior are associated with a higher quality of life and lower mortality rates for cancer survivors, a growing population group. Studies detailing the behavior of cancer survivors are limited. Therefore, we investigated physical activity and sedentary behavior of cancer survivors using data from the National Health and Nutrition Examination Survey (NHANES) 2007-2010. Participants were those who provided physical activity and sedentary behavior data. Those who were pregnant, <20 years old, or <3 years from their cancer diagnosis were excluded. A cancer case was a self-reported diagnosis by a physician. We identified 741 cancer survivors and 10,472 non-cancer participants. After adjustment for age, race, gender, education status, body mass index, and smoking status, cancer survivors (n = 10,472) reported significantly longer duration of sedentary behavior (OR = 1.42, 95% CI (1.12, 1.80) for 8 or more hours, p-value for trend = 0.09), compared to non-cancer participants (n = 741). They also reported non-significant increases in maximum intensity, duration, frequency, and energy expenditure, whereas they reported significant increases in moderate intensity (OR = 1.26, 95% CI (1.01, 1.57)), moderate frequency (1-4 times/week) (OR = 1.32, 95% CI (1.00, 1.74)), and moderate energy expenditure (4018.5-7623.5 kcal) (OR = 1.30, 95% CI (1.00, 1.71)) of physical activity, compared to non-cancer participants. These patterns are similar for breast and prostate cancer survivors, with prostate cancer survivors more likely to engage in physical activity for more than one hour per day (OR = 1.98, 95% CI (1.05, 3.71)). Our findings suggest that cancer survivors tend to have more physical activity, but they are also more likely to engage in sedentary behavior.

In situ vaccination with CD204 gene-silenced dendritic cell, not unmodified dendritic cell, enhances radiation therapy of prostate cancer.

Given the complexity of prostate cancer progression and metastasis, multimodalities that target different aspects of tumor biology, for example, radiotherapy in conjunction with immunotherapy, may provide the best opportunities for promoting clinical benefits in patients with high-risk localized prostate cancer. Here, we show that intratumoral administration of unmodified dendritic cells (DC) failed to synergize with fractionated radiotherapy. However, ionizing radiation combined with in situ vaccination with DCs, in which the immunosuppressive scavenger receptor A (SRA/CD204) has been downregulated by lentivirus-mediated gene silencing, profoundly suppressed the growth of two mouse prostate cancers (e.g., RM1 and TRAMP-C2) and prolonged the lifespan of tumor-bearing animals. Treatment of subcutaneous tumors with this novel combinatorial radioimmunotherapeutic regimen resulted in a significant reduction in distant experimental metastases. SRA/CD204-silenced DCs were highly efficient in generating antigen or tumor-specific T cells with increased effector functions (e.g., cytokine production and tumoricidal activity). SRA/CD204 silencing-enhanced tumor cell death was associated with elevated IFN-γ levels in tumor tissue and increased tumor-infiltrating CD8(+) cells. IFN-γ neutralization or depletion of CD8(+) cells abrogated the SRA/CD204 downregulation-promoted antitumor efficacy, indicating a critical role of IFN-γ-producing CD8(+) T cells. Therefore, blocking SRA/CD204 activity significantly enhances the therapeutic potency of local radiotherapy combined with in situ DC vaccination by promoting a robust systemic antitumor immunity. Further studies are warranted to test this novel combinatorial approach for translating into improved clinical outcomes in patients with prostate cancer.

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, N(G)-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.

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).