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At least 901 records · Page 50
All sourcesNASA NTRSDOE OSTI
DOE OSTI2011

Adenovirus E2F1 Overexpression Sensitizes LNCaP and PC3 Prostate Tumor Cells to Radiation In Vivo

Purpose: We previously showed that E2F1 overexpression radiosensitizes prostate cancer cells in vitro. Here, we demonstrate the radiosensitization efficacy of adenovirus (Ad)-E2F1 infection in growing (orthotopic) LNCaP and (subcutaneous) PC3 nude mice xenograft tumors. Methods and Materials: Ad-E2F1 was injected intratumorally in LNCaP (3 x 10{sup 8} plaque-forming units [PFU]) and PC3 (5 x 10{sup 8} PFU) tumors treated with or without radiation. LNCaP tumor volumes (TV) were measured by magnetic resonance imaging, caliper were used to measure PC3 tumors, and serum prostate-specific antigen (PSA) levels were determined by enzyme-linked immunosorbent assay. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and key proteins involved in cell death signaling were analyzed by Western blotting. Results: Intracellular overexpression of Ad-E2F1 had a significant effect on the regression of TV and reduction of PSA levels relative to that of adenoviral luciferase (Ad-Luc)-infected control. The in vivo regressing effect of Ad-E2F1 on LNCaP tumor growth was significant (PSA, 34 ng/ml; TV, 142 mm{sup 3}) compared to that of Ad-Luc control (PSA, 59 ng/ml; TV, 218 mm{sup 3}; p <0.05). This effect was significantly enhanced by radiation therapy (compare: Ad-E2F1+RT/PSA, 16 ng/ml, and TV, 55 mm{sup 3} to Ad-Luc+RT/PSA, 42 ng/ml, and TV, 174 mm{sup 3}, respectively; p <0.05). For PC3 tumors, the greatest effect was observed with Ad-E2F1 infection alone; there was little or no effect when radiotherapy (RT) was combined. However, addition of RT enhanced the level of in situ apoptosis in PC3 tumors. Molecularly, addition of Ad-E2F1 in a combination treatment abrogated radiation-induced BCL-2 protein expression and was associated with an increase in activated BAX, and together they caused a potent radiosensitizing effect, irrespective of p53 and androgen receptor functional status. Conclusions: We show here for the first time that ectopic overexpression of E2F1 in vivo, using an adenoviral vector, significantly inhibits orthotopic p53 wild-type LNCaP tumors and subcutaneous p53-null PC3 tumors in nude mice. Furthermore, we demonstrate that E2F1 strongly sensitizes LNCaP tumors to RT. These findings suggest that E2F1 overexpression can sensitize prostate tumor cells in vivo, independent of p53 or androgen receptor status.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Widespread Dependence of Backup NHEJ on Growth State: Ramifications for the Use of DNA-PK Inhibitors

Purpose: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4{sup -/-} mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. Methods and Materials: DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of {gamma}-H2AX foci, at different stages of growth. Results: Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by {gamma}-H2AX foci analysis, and leads to enhanced cell killing. Conclusions: These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2011

Tocopherol Succinate: Modulation of Antioxidant Enzymes and Oncogene Expression, and Hematopoietic Recovery

Purpose: A class of naturally occurring isoforms of tocopherol (tocols) was shown to have varying degrees of protection when administered before radiation exposure. We recently demonstrated that {alpha}-tocopherol succinate (TS) is a potential radiation prophylactic agent. Our objective in this study was to further investigate the mechanism of action of TS in mice exposed to {sup 60}Co {gamma}-radiation. Methods and Materials: We evaluated the effects of TS on expression of antioxidant enzymes and oncogenes by quantitative RT-PCR in bone marrow cells of {sup 60}Co {gamma}-irradiated mice. Further, we tested the ability of TS to rescue and repopulate hematopoietic stem cells by analyzing bone marrow cellularity and spleen colony forming unit in spleen of TS-injected and irradiated mice. Results: Our results demonstrate that TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice at different time points. TS also increased colony forming unit-spleen numbers and bone marrow cellularity in irradiated mice. Conclusions: Results provide additional support for the observed radioprotective efficacy of TS and insight into mechanisms.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2011

Radiation Pneumopathy in the Rat After Intravenous Application of {sup 188}Re-Labeled Microspheres

Purpose: To determine the dose dependence and kinetics of pneumopathy after systemic administration of rhenium-188 ({sup 188}Re)-labeled microspheres in a rat model. Methods and Materials: {sup 188}Re-microspheres were injected intravenously into adult Wistar rats (n = 54, age, 8 {+-} 2 months). The rats were divided into 6 groups according to the intended absorbed dose in the lung (maximum 60 Gy). Gamma camera scans were used to estimate the individual whole lung doses. One control group (n = 5) received nonlabeled microspheres. The breathing rate was measured before and weekly after the treatment using whole body plethysmography until 24 weeks. An increase in the breathing rate by 20% compared with the individual pretreatment control value was defined as the quantal endpoint for dose-effect analyses. Results: A biphasic increase in the breathing rate was observed. The first impairment of lung function occurred in Weeks 3-6. For late changes, the interval to onset was clearly dose dependent and was 17 weeks (10-30 Gy) and 10 weeks (50-60 Gy), respectively. The incidence of the response was highly dependent on the estimated lung dose. The median effective dose for an early and late response was virtually identical (19.9 {+-} 0.6 Gy and 20.4 {+-} 3.1 Gy, respectively). A significant correlation was found between the occurrence of an early and a late effect in the same rat, suggesting a strong consequential component. Conclusions: The effects of radiolabeled microspheres can be studied longitudinally in a rat model, using changes in the breathing rate as the functional, clinically relevant response. The isoeffective doses from the present study using radionuclide administration and those from published investigations of homogeneous external beam radiotherapy are almost similar.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Reduction of Radiation-Induced Vascular Nitrosative Stress by the Vitamin E Analog {gamma}-Tocotrienol: Evidence of a Role for Tetrahydrobiopterin

Purpose: The vitamin E analog {gamma}-tocotrienol (GT3) is a powerful radioprotector. GT3 reduces postradiation vascular peroxynitrite production, an effect dependent on inhibition of hydroxy-methylglutaryl-coenzyme A reductase. Hydroxy-methylglutaryl-coenzyme A reductase inhibitors mediate their pleiotropic effects via endothelial nitric oxide synthase that requires the cofactor tetrahydrobiopterin (BH4). This study investigated the effects of radiation on BH4 bioavailability and of GT3 on BH4 metabolism. Methods and Materials: Mice were exposed to 8.5 Gy of total body irradiation (TBI). Lung BH4 and total biopterin concentrations were measured 0, 3.5, 7, 14, and 21 days after TBI by use of differential oxidation followed by high-performance liquid chromatography. The effect of exogenous GT3 and BH4 treatment on postradiation vascular oxidative stress and bone marrow colony-forming units were assessed in vivo. The effect of GT3 on endothelial cell apoptosis and endothelial expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GTPCH), GTPCH feedback regulatory protein (GFRP), GFRP transcription, GFRP protein levels, and GFRP-GTPCH protein binding was determined in vitro. Results: Compared with baseline levels, lung BH4 concentrations decreased by 24% at 3.5 days after TBI, an effect that was reversed by GT3. At 14 and 21 days after TBI, compensatory increases in BH4 (58% and 80%, respectively) were observed. Relative to vehicle-treated controls, both GT3 and BH4 supplementation reduced postirradiation vascular peroxynitrite production at 3.5 days (by 66% and 33%, respectively), and BH4 resulted in a 68% increase in bone marrow colony-forming units. GT3 ameliorated endothelial cell apoptosis and reduced endothelial GFRP protein levels and GFRP-GTPCH binding by decreasing transcription of the GFRP gene. Conclusions: BH4 bioavailability is reduced in the early postradiation phase. Exogenous administration of BH4 reduces postirradiation vascular oxidative stress. GT3 potently reduces the expression of GFRP, one of the key regulatory proteins in the BH4 pathway, and may thus exert some of its beneficial effects on postradiation free radical production partly by counteracting the decrease in BH4.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Altered Cross-Linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27-Mediated Radioresistance

Purpose: HSP27 or HSP25 negatively regulates apoptosis pathways after radiation or chemotherapeutic agents. Abrogation of HSP27 function may be a candidate target for overcoming radio- and chemoresistance. Methods and Materials: Zerumbone (ZER), a cytotoxic component isolated from Zingiber zerumbet smith. Clonogenic survival assay and flow cytometry after Annexin V staining were performed to determine in vitro sensitization effects of ZER with ionizing radiation. A nude mouse xenografting system was also applied to detect in vivo radiosensitizing effects of ZER. Results: ZER produced cross-linking of HSP27, which was dependent on inhibition of the monomeric form of HSP27. ZER was directly inserted between the disulfide bond in the HSP27 dimer and modified normal HSP27 dimerization. Pretreatment with ZER before radiation inhibited the binding affinity between HSP27 and apoptotic molecules, such as cytochrome c and PKC{delta}, and induced sensitization in vitro and in an in vivo xenografted nude mouse system. Structural analogs lacking only the carbonyl group in ZER, such as {alpha}-humulene (HUM) and 8-hydroxy-humulen (8-OH-HUM), did not affect normal cross-linking of HSP27 and did not induce radiosensitization. Conclusions: We suggest that altered cross-linking of HSP27 by ZER is a good strategy for abolishing HSP27-mediated resistance.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2011

Spinal Cord Tolerance in the Age of Spinal Radiosurgery: Lessons From Preclinical Studies

Clinical implementation of spinal radiosurgery has increased rapidly in recent years, but little is known regarding human spinal cord tolerance to single-fraction irradiation. In contrast, preclinical studies in single-fraction spinal cord tolerance have been ongoing since the 1970s. The influences of field length, dose rate, inhomogeneous dose distributions, and reirradiation have all been investigated. This review summarizes literature regarding single-fraction spinal cord tolerance in preclinical models with an emphasis on practical clinical significance. The outcomes of studies that incorporate uniform irradiation are surprisingly consistent among multiple small- and large-animal models. Extensive investigation of inhomogeneous dose distributions in the rat has demonstrated a significant dose-volume effect while preliminary results from one pig study are contradictory. Preclinical spinal cord dose-volume studies indicate that dose distribution is more critical than the volume irradiated suggesting that neither dose-volume histogram analysis nor absolute volume constraints are effective in predicting complications. Reirradiation data are sparse, but results from guinea pig, rat, and pig studies are consistent with the hypothesis that the spinal cord possesses a large capacity for repair. The mechanisms behind the phenomena observed in spinal cord studies are not readily explained and the ability of dose response models to predict outcomes is variable underscoring the need for further investigation. Animal studies provide insight into the phenomena and mechanisms of radiosensitivity but the true significance of animal studies can only be discovered through clinical trials.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Interstitial Fluid Pressure and Vascularity of Intradermal and Intramuscular Human Tumor Xenografts

Purpose: High interstitial fluid pressure (IFP) in tumors has been shown to be associated with poor prognosis. Mechanisms underlying the intertumor heterogeneity in IFP were investigated in this study. Methods and Materials: A-07 melanoma xenografts were transplanted intradermally or intramuscularly in BALB/c nu/nu mice. IFP was measured in the center of the tumors with a Millar catheter. Tumor blood perfusion and extracellular volume fraction were assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). The necrotic fraction, vascular density, and vessel diameters of the tumors were determined by image analysis of histological preparations. Results: Significant intertumor heterogeneity in IFP, blood perfusion, and microvascular morphology was observed whether the tumors were transplanted intradermally or intramuscularly. High IFP was mainly a consequence of high resistance to blood flow caused by low vessel diameters in either transplantation site. IFP decreased with increasing blood perfusion in intradermal tumors and increased with increasing blood perfusion in intramuscular tumors, mainly because the morphology of the tumor microvasculature differed systematically between the two tumor models. Conclusion: The potential of DCE-MRI as a noninvasive method for assessing the IFP of tumors may be limited because any relationship between IFP and blood perfusion may differ with the tumor growth site.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

In Vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated With Therapeutic Outcome

Purpose: To develop a combination treatment consisting of combretastatin A-4-phosphate (CA4P) with radiation based on tumor oxygenation status. Methods and Materials: In vivo near-infrared spectroscopy (NIRS) and diffusion-weighted (DW) magnetic resonance imaging (MRI) were applied to noninvasively monitor changes in tumor blood oxygenation and necrosis induced by CA4P (30 mg/kg) in rat mammary 13762NF adenocarcinoma, and the evidence was used to optimize combinations of CA4P and radiation treatment (a single dose of 5 Gy). Results: NIRS showed decreasing concentrations of tumor vascular oxyhemoglobin and total hemoglobin during the first 2 h after CA4P treatment, indicating significant reductions in tumor blood oxygenation and perfusion levels (p < 0.001). Twenty-four hours later, in response to oxygen inhalation, significant recovery was observed in tumor vascular and tissue oxygenation according to NIRS and pimonidazole staining results, respectively (p < 0.05). DW MRI revealed significantly increased water diffusion in tumors measured by apparent diffusion coefficient at 24 h (p < 0.05), suggesting that CA4P-induced central necrosis. In concordance with the observed tumor oxygen dynamics, we found that treatment efficacy depended on the timing of the combined therapy. The most significant delay in tumor growth was seen in the group of tumors treated with radiation while the rats breathed oxygen 24 h after CA4P administration. Conclusions: Noninvasive evaluation of tumor oxygen dynamics allowed us to rationally enhance the response of syngeneic rat breast tumors to combined treatment of CA4P with radiation.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Long-Term Effects of {sup 56}Fe Irradiation on Spatial Memory of Mice: Role of Sex and Apolipoprotein E Isoform

Purpose: To assess whether the effects of cranial {sup 56}Fe irradiation on the spatial memory of mice in the water maze are sex and apolipoprotein E (apoE) isoform dependent and whether radiation-induced changes in spatial memory are associated with changes in the dendritic marker microtubule-associated protein 2 (MAP-2) and the presynaptic marker synaptophysin. Methods and Materials: Two-month-old male and female mice expressing human apoE3 or apoE4 received either a 3-Gy dose of cranial {sup 56}Fe irradiation (600 MeV/amu) or sham irradiation. Mice were tested in a water maze task 13 months later to assess effects of irradiation on spatial memory retention. After behavioral testing, the brain tissues of these mice were analyzed for synaptophysin and MAP-2 immunoreactivity. Results: After irradiation, spatial memory retention of apoE3 female, but not male, mice was impaired. A general genotype deficit in spatial memory was observed in sham-irradiated apoE4 mice. Strikingly, irradiation prevented this genotype deficit in apoE4 male mice. A similar but nonsignificant trend was observed in apoE4 female mice. Although there was no change in MAP-2 immunoreactivity after irradiation, synaptophysin immunoreactivity was increased in irradiated female mice, independent of genotype. Conclusions: The effects of {sup 56}Fe irradiation on the spatial memory retention of mice are critically influenced by sex, and the direction of these effects is influenced by apoE isoform. Although in female mice synaptophysin immunoreactivity provides a sensitive marker for effects of irradiation, it cannot explain the apoE genotype-dependent effects of irradiation on the spatial memory retention of the mice.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Outcome and Prognostic Factors of Radiation Therapy for Medulloblastoma

Purpose: To investigate treatment outcome and prognostic factors after radiation therapy in patients with medulloblastomas (MB). Methods and Materials: Sixty-six patients with histologically confirmed MB were treated at University Hospital of Heidelberg between 1985 and 2009. Forty-two patients (64%) were pediatric ({<=}18 years), and 24 patients (36%) were adults. Tumor resection was performed in all patients and was complete in 47%. All patients underwent postoperative craniospinal irradiation (CSI) delivering a median craniospinal dose of 35.5 Gy with additional boosts to the posterior fossa up to 54.0 Gy. Forty-seven patients received chemotherapy, including 21 in whom chemotherapy was administered before CSI. Statistical analysis was performed using the log-rank test and the Kaplan-Meier method. Results: Median follow-up was 93 months. Overall survival (OS) and local and distant progression-free survival (LPFS and DPFS) were 73%, 62%, and 77% at 60 months. Both local and distant recurrence predisposed for significantly reduced OS. Macroscopic complete tumor resection, desmoplastic histology and early initiation of postoperative radiation therapy within 28 days were associated with improved outcome. The addition of chemotherapy did not improve survival rates. Toxicity was moderate. Conclusions: Complete resection of MB followed by CSI yields long survival rates in both children and adults. Delayed initiation of CSI is associated with poor outcome. Desmoplastic histology is associated with improved survival. The role of chemotherapy, especially in the adult population, must be further investigated in clinical studies.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Two Strategies for the Development of Mitochondrion-Targeted Small Molecule Radiation Damage Mitigators

Purpose: To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules. Methods and Materials: We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53{sup +/+} and p53{sup -/-} murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI). Results: Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53{sup +/+} (p = 0.0396 and p = 0.0071, respectively) and p53{sup -/-} cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53{sup -/-} cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively). Conclusion: Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Fractionated External Beam Radiotherapy as a Suitable Preparative Regimen for Hepatocyte Transplantation After Partial Hepatectomy

Purpose: Hepatocyte transplantation is strongly considered to be a promising option to correct chronic liver failure through repopulation of the diseased organ. We already reported on extensive liver repopulation by hepatocytes transplanted into rats preconditioned with 25-Gy single dose selective external beam irradiation (IR). Herein, we tested lower radiation doses and fractionated protocols, which would be applicable in clinical use. Methods and Material: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with partial liver external beam single dose IR at 25 Gy, 8 Gy, or 5 Gy, or fractionated IR at 5 x 5 Gy or 5 x 2 Gy. Four days after completion of IR, a partial hepatectomy (PH) was performed to resect the untreated liver section. Subsequently, 12 million wild-type (DPPIV{sup +}) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor cell integration and liver repopulation was studied 16 weeks after transplantation by means of immunofluorescence and DPPIV-luminescence assay. Results: Donor hepatocyte integration and liver repopulation were more effective in the irradiated livers following pretreatment with the IR doses 1 x 25 Gy and 5 x 5 Gy (formation of large DPPIV-positive cell clusters) than single-dose irradiation at 8 Gy or 5 Gy (DPPIV-positive clusters noticeably smaller and less frequent). Quantitative analysis of extracted DPPIV revealed signals exceeding the control level in all transplanted animals treated with IR and PH. Compared with the standard treatment of 1 x 25 Gy, fractionation with 5 x 5 Gy was equally efficacious, the Mann-Whitney U test disclosing no statistically significant difference (p = 0.146). The lower doses of 1 x 5 Gy, 1 x 8 Gy, and 5 x 2 Gy were significantly less effective with p < 0.05. Conclusion: This study suggests that fractionated radiotherapy in combination with PH is a conceivable pretreatment approach to prime the host liver for hepatocyte transplantation, thus bringing the experimental model a step closer to clinical application.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

Volume-Dependent Expression of In-Field and Out-of-Field Effects in the Proton-Irradiated Rat Lung

Purpose: To investigate whether occurance of early radiation effects in lung tissue depends on local dose only. Methods and Materials: Twenty-five percent, 50%, 66%, 88%, or 100% of the rat lung was irradiated using single fractions of 150-MeV protons. For all volumes, in-field and out-of-field dose-response curves were obtained 8 weeks after irradiation. The pathohistology of parenchymal inflammation, infiltrates, fibrosis, and vascular damage and the relative expression of proinflammatory cytokines interleukin (IL)-1{alpha}, transforming growth factor-{beta}, IL-6, and tumor necrosis factor-{alpha} were assessed. Results: For all histologic endpoints, irradiated dose- and volume-dependent in-field and out-of-field effects were observed, albeit with different dynamics. Of note, the out-of-field effects for vascular damage were very similar to the in-field effects. Interestingly, only IL-6 showed a clear dose-dependent increase in expression both in-field and out-of-field, whereas the expression levels of IL-1{alpha}, transforming growth factor-{beta}, and tumor necrosis factor-{alpha} were either very low or without a clear dose-volume relation. As such, none of the radiation effects studied depended only on local dose to the tissue. Conclusion: The effects of radiation to lung tissue do not only depend on local dose to that tissue. Especially at high-volume irradiation, lung damage seems to present globally rather than locally. The accuracy of predictive modeling may be improved by including nonlocal effects.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

The Role of Platelet Factor 4 in Radiation-Induced Thrombocytopenia

Purpose: Factors affecting the severity of radiation-induced thrombocytopenia (RIT) are not well described. We address whether platelet factor 4 (PF4; a negative paracrine for megakaryopoiesis) affects platelet recovery postradiation. Methods and Materials: Using conditioned media from irradiated bone marrow (BM) cells from transgenic mice overexpressing human (h) PF4 (hPF4+), megakaryocyte colony formation was assessed in the presence of this conditioned media and PF4 blocking agents. In a model of radiation-induced thrombocytopenia, irradiated mice with varying PF4 expression levels were treated with anti-hPF4 and/or thrombopoietin (TPO), and platelet count recovery and survival were examined. Results: Conditioned media from irradiated BM from hPF4+ mice inhibited megakaryocyte colony formation, suggesting that PF4 is a negative paracrine released in RIT. Blocking with an anti-hPF4 antibody restored colony formation of BM grown in the presence of hPF4+ irradiated media, as did antibodies that block the megakaryocyte receptor for PF4, low-density lipoprotein receptor-related protein 1 (LRP1). Irradiated PF4 knockout mice had higher nadir platelet counts than irradiated hPF4+/knockout litter mates (651 vs. 328 x 106/mcL, p = 0.02) and recovered earlier (15 days vs. 22 days, respectively, p <0.02). When irradiated hPF4+ mice were treated with anti-hPF4 antibody and/or TPO, they showed less severe thrombocytopenia than untreated mice, with improved survival and time to platelet recovery, but no additive effect was seen. Conclusions: Our studies show that in RIT, damaged megakaryocytes release PF4 locally, inhibiting platelet recovery. Blocking PF4 enhances recovery while released PF4 from megakaryocytes limits TPO efficacy, potentially because of increased release of PF4 stimulated by TPO. The clinical value of blocking this negative paracrine pathway post-RIT remains to be determined.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Assessment of Tumor Radioresponsiveness and Metastatic Potential by Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Purpose: It has been suggested that gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may provide clinically useful biomarkers for personalized cancer treatment. In this preclinical study, we investigated the potential of DCE-MRI as a noninvasive method for assessing the radioresponsiveness and metastatic potential of tumors. Methods and Materials: R-18 melanoma xenografts growing in BALB/c nu/nu mice were used as experimental tumor models. Fifty tumors were subjected to DCE-MRI, and parametric images of K{sup trans} (the volume transfer constant of Gd-DTPA) and v{sub e} (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. The tumors were irradiated after the DCE-MRI, either with a single dose of 10 Gy for detection of radiobiological hypoxia (30 tumors) or with five fractions of 4 Gy in 48 h for assessment of radioresponsiveness (20 tumors). The host mice were then euthanized and examined for lymph node metastases, and the primary tumors were resected for measurement of cell survival in vitro. Results: Tumors with hypoxic cells showed significantly lower K{sup trans} values than tumors without significant hypoxia (p < 0.0001, n = 30), and K{sup trans} decreased with increasing cell surviving fraction for tumors given fractionated radiation treatment (p < 0.0001, n = 20). Tumors in metastasis-positive mice had significantly lower K{sup trans} values than tumors in metastasis-negative mice (p < 0.0001, n = 50). Significant correlations between v{sub e} and tumor hypoxia, radioresponsiveness, or metastatic potential could not be detected. Conclusions: R-18 tumors with low K{sup trans} values are likely to be resistant to radiation treatment and have a high probability of developing lymph node metastases. The general validity of these observations should be investigated further by studying preclinical tumor models with biological properties different from those of the R-18 tumors.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2010

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

To clarify the involvement of signaling of transforming growth factor (TGF)-{beta} during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6 weeks after starting promotion) and late-stage promotion (57 weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P{sup +} foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P{sup +} lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P{sup +} foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P{sup -} foci induced by promotion with agonists of peroxisome proliferator-activated receptor-{alpha} did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P{sup +} proliferative lesions.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2010

Temporal study of acetaminophen (APAP) and S-adenosyl-L-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity

Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 g) were treated with vehicle (Veh; water 15 ml/kg ip injections), 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered 1 h after APAP injection (SAMe and SAMe + APAP). Hepatic tissue was collected 2, 4, and 6 h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4 h by APAP overdose, but not at 2 or 6 h. APAP depressed mitochondrial SAMe levels at 4 and 6 h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4 h following APAP administration. SAMe administration following APAP (SAMe + APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗