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PURPOSE/OBJECTIVE(S): hypoxia has been commonly observed in human nasopharyngeal carcinoma (NPC) which is associated with resistance to radiation and malignant phenotypes. The purpose of the study was to apply our build-in hypoxia-driven dual reporter model to explore the effect of hypoxia on radiosensitivity in vitro and in vivo. MATERIALS/METHODS The modalities to research the effect of hypoxia in vitro includes clonogenic survival assay and γH2AX foci formation. Tumor growth delay with radiation and/or cisplatin, imaging study on spatial biodistribution between blood perfusion (Hoechst 33342), endogenous (eGFP and CA9) and exogenous (pimonidazole) hypoxia markers were used to investigate the effect of hypoxia in vivo. RESULTS The constructed hypoxia-driven dual-reporter model contains 9 repeats of hypoxia responsive element and fusion gene of 9xHRE-HSV1-TKeGFP in NPC cells (abbreviated as SUNE-1/HRE cells) in which hypoxia-induced molecular events can be visualized by fluorescence or nuclear imaging techniques. There is no difference in radiosensitivity between SUNE1/Parental and SUNE1/HRE cells, while cisplatin significantly increases radiosensitivity in both cell lines. γH2AX serves as a surrogate for unrepaired DSB. When the cells are exposed to 0.5% O2, eGFP is observed only in SUNE1/HRE cells. DSB repair rate under hypoxia is similar in SUNE1/Parental and SUNE1/HRE cells. The number of γH2AX foci is dramatically higher in the presence of cisplatin compared to that given irradiation alone under the condition of normoxia or hypoxia. It indicates that the addition of cisplatin enhances radiosensitivity by compromising the ability of cells to repair DSB. Cisplatin alone shows modest whereas irradiation moderate effect on delayed tumor growth. Combined treatment of irradiation and cisplatin further enhances the effect on tumor growth delay. High expression of eGFP is observed in the nonperfused regions. The spatial biodistribution of hypoxia-induced expression of eGFP reporter gene is similar to other endogenous or exogenous hypoxia biomarkers, i.e., CA9 and pimonidazole. CONCLUSION The build-in hypoxia-driven dual reporter 9HRE-HSV1-TKeGFP hypoxia research model does not modify intrinsic radiosensitivity of SUNE1 cells under the condition of normoxia or hypoxia, suggesting it be used as preclinical model to explore the therapeutic efficacy of various treatment modalities to overcome hypoxia by fluorescence or nuclear imaging techniques.

Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1α in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.

Purpose: To develop a new method for accurate measurement of dynamic respiratory tidal volume, we investigate the feasibility of measuring torso volume change using optical surfaceimaging (OSI). Methods: Based on a validated volume conservation theory, the tidal volume is equal to the volume change of the torso during quiet respiration (Li et al, PMB, 54:1693, 2009). A clinical OSI system was employed to acquire surfaceimages of seven geometric phantoms and two ‘deformable’ torso phantoms. The mesh surfaceimages were converted into contours for volume calculation using a treatment planning system. For geometric phantoms, their volumes under the incomplete surfaceimages were calculated with aid of their symmetry. The results were compared with theoretical calculation and water containment experiments. For deformable torso phantoms, we created volume‐controlled deformation stages by placing deformable PlayDoh (DPD) materials on top of rigid Rando/Thorax phantoms, mimicking respiration‐induced torso surface elevation and volume change. The volume difference under the surfaces with and without the DPD padding was calculated with aid of a common posterior line to enclose the region of interest. Three different volumes of DPD padding (>500cc) were mounted on the torso phantoms and CT scanned for volume measurements. Results: For geometric phantoms, the OSI measured volume had accuracy (±1s) of 0.0%±1.6% (vs. geometric volume calculation) and 0.6%±3.8% (vs. water containment experiment). For deformable torso phantoms, the volume change was measured using OSI with an accuracy of 1.5%±2.5% against the measured volume using CTimaging.Linear regression showed a one‐to‐one relationship between the OSI volumes and CT volumes with a slope of 1.003 (r2=0.999). Conclusions: The optical surfaceimaging system can accurately measure the volume of geometric phantoms and the volume change of deformable torso phantoms. The accuracy is about 3% against standard volume measurement methods. Further study on human subjects is under investigation. Memorial Sloan‐Kettering Cancer Center has a reserach agreement with Vision RT, Inc.

The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissue, may provide an opportunity for cancer-specific gene therapy. The purpose of the present study was to investigate whether hypoxia-driven triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to ganciclovir (GCV) and 5-fluorocytosine (5-FC), and sensitizes human colorectal cancer to radiation in vitro and in vivo. Stable transfectant of human colorectal HCT8 cells was established which expressed hypoxia-inducible vectors (HRE-TK/eGFP and HRE-CD/UPRT/mDsRed). Hypoxia-induced expression/function of TK, CD and UPRT was verified by western blot analysis, flow cytometry, fluorescent microscopy and cytotoxicity assay of GCV and 5-FC. Significant radiosensitization effects were detected after 5-FC and GCV treatments under hypoxic conditions. In the tumor xenografts, the distribution of TK/eGFP and CD/UPRT/mDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC and GCV in mice in combination with local irradiation resulted in tumor regression, as compared with prodrug or radiation treatments alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple suicide gene therapy may have the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

Previously, we found that extremely low frequency (ELF) electric fields were able to elicit an approximate 3.5-fold increase in heat shock gene expression, a response which may have applicability to cancer therapy. Based on recent studies demonstrating the ability of magnetic fields to influence gene expression, we hypothesized that low level static magnetic fields may be able to affect heat shock gene expression while avoiding some of the clinical difficulties that arise with electric fields. Transfected rat primary cells in monolayer were exposed to magnetic fields of 1 to 440 mT for 16, 24, or 48 h starting at 24 and 48 h post transfection. Heat shock protein (HSP70) expression, as indicated by a promoter linked luciferase reporter, was followed for up to 96 h and showed a dependence on flux density, exposure duration, and start time post transfection. A nonlinear response was observed for increasing flux density with a maximum of a 3.5-fold increase in expression for 48 h of exposure starting 48 h after transfection. These results demonstrate an enhancement of gene expression similar in magnitude to that observed with external electric field exposure, while eliminating many of the clinical complications. Bioelectromagnetics. 35:406–413, 2014. © 2014 Wiley Periodicals, Inc.

Gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, has shown promise in clinical trials. In this preclinical study using stable cell lines and xenograft tumor models, we show that a triple-suicide-gene GDEPT approach produce enhanced therapeutic efficacy over previous methods. Importantly all the three genes (thymidine kinase, cytosine deaminase, and uracil phosphoribosyltransferase) function simultaneously as effectors for GDEPT and markers for multimodality molecular imaging (MMI), using positron-emission-tomography (PET), magnetic resonance-spectroscopy (MRS), and optical (fluorescent and bioluminescent) techniques. It was demonstrated that MMI can evaluate the distribution and function/activity of the triple-suicide-gene. The concomitant expression of these genes significantly enhances prodrug cytotoxicity and radiosensitivity in vitro and in vivo.

// Jun Dong 1, * , Tian Zhang 1, * , Yufeng Ren 1, * , Zhenyu Wang 1, * , Clifton C. Ling 2 , Fuqiu He 2 , Gloria C. Li 2 , Chengtao Wang 1 , Bixiu Wen 1, 2 1 Department of Radiation Oncology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China 2 Department of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA * Co-first author Correspondence to: Bixiu Wen, email: wenbix@mail.sysu.edu.cn Chengtao Wang, email: wangcht5@mail.sysu.edu.cn Keywords: DNA-PKcs, non-homologous end-joining, double-strand break, NU7441, nasopharyngeal carcinoma Received: September 08, 2016 Accepted: January 25, 2017 Published: February 07, 2017 ABSTRACT DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase. Inhibition of DNA-PKcs prolonged IR-induced G2/M phase arrest because of sequential activation of cell cycle checkpoints. DSBs were introduced, and cell cycle checkpoints were recruited after exposure to IR in nasopharyngeal carcinoma SUNE-1 cells. NU7441 radiosensitized MEF cells and SUNE-1 cells by interfering with DSB repair. Together, these results reveal a mechanism in which coupling of DSB repair with the cell cycle radiosensitizes NHEJ repair-deficient cells, justifying further development of DNA-PK inhibitors in cancer therapy.

To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy.Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy.Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone.Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

Purpose To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. Methods and Materials Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. Results Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was ∼1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2–1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. Conclusion Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

Clinically achievable mild temperature local hyperthermia (43 degrees C) has been demonstrated to be an effective adjuvant to radiotherapy in pre-clinical and clinical studies. In this article, we briefly review the recent progress in the following areas: (1) the effect of mild temperature hyperthermia (MTH) on tumour hypoxia and blood perfusion as assessed by dual marker immunohistochemistry (IHC); (2) the kinetics of MTH induced changes in tumour hypoxia; (3) the potential role of heat-induced tumour reoxygenation on radio- and chemo-sensitisation; (4) the potential role of MTH in combination with vascular targeting agents (VTAs) on tumour response; and (5) non-invasive detection of changes in tumour oxygenation and blood perfusion. It is shown that MTH, by itself or in combination with VTAs, leads to changes in tumour perfusion and oxygenation with potential for radio- and chemo-sensitisation.

To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques.HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5. Pimonidazole was given 1h preheating, EF5 at various times during or after treatment, 1h later the animals were sacrificed. Blood vessels were identified by CD31 staining, and perfusion by Hoechst 33342 injected 1 min pre-sacrifice.The overall hypoxic fraction was significantly decreased by MTH during and immediately after heating. However, MTH induced both increases and decreases in tumor hypoxia in different parts of the tumor. Specifically, MTH decreased hypoxia in the regions with relatively well-perfused blood vessels, but increased hypoxia in regions that were poorly perfused. At 24-h post heating, newly formed hypoxic regions surrounded previously-hypoxic foci, which in turn surrounded pimonidazole-stained debris. Quantitative analysis did not evince changes in tumor oxygenation due to MTH at 24h post-treatment.In this xenograft model, the effect of MTH on tumor oxygenation was variable, both spatially and kinetically. Overall tumor oxygenation was improved during and after heating, but the effect was short-lived.

In this study, we investigated whether the inhibition o fe ndogenous phosphorylation o fm itogen-activated protein kinase (MAPK) and diallyl disulfide (DADS)-induced phosphorylation of MAPKs with MAPK specific inhibitors, SB203580 and U0126 (for phospho-p38 and phospho-p42/p44, respectively), can induce or enhance apoptosis in human CNE2 nasopharyngeal carcinoma cells. Our data demonstrate that MAPK inhibitors decrease the viability of CNE2 cells, stimulate typical apoptotic morphologic changes, and enhance DADS-induced apoptosis. The present findings indicate that phosphorylation of MAPKs plays an important cytoprotective role in CNE2 cell apoptosis and the DADS-induced apoptotic process.

Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function.Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy ((19)F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts.Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor (19)F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes.This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings.

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH2-terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA end-binding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of γ-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy. [Cancer Res 2007;67(2):634–42]

This study was undertaken to elucidate the effect of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, in suppressing human nasopharyngeal carcinoma cells. A potent increase (of at least 9-fold) in apoptotic cells has accompanied 1) a decrease in cell viability, 2) a increase of the fraction of S-phase cells by up to 63.8%, and 3) a transient increase of the phospho-p38 and phospho-p42/44 (phosphorylated p38 MAPK and phosphorylated p42/44 MAPK) in a time- and concentration-dependent manner. These results indicate that DADS can induce apoptosis in human nasopharyngeal carcinoma cells via, at least partly, S-phase block of the cell cycle, related to a rise in MAPK phosphorylation.

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent. Microhomology-mediated end joining (MHEJ) is always accompanied by a deletion that spans one of the two homologous sequences and the intervening sequence, if any. In this study we evaluated several factors affecting the relative contribution of MHEJ to DNA end joining using nuclear extracts and DNA substrates containing 10-bp repeats at the ends. We found that the occurrence of MHEJ is determined by the relative abundance of nuclear proteins. At low DNA/protein ratios, an error-free end-joining mechanism predominated over MHEJ. As the DNA/protein ratio increased, MHEJ became predominant. We show that the nuclear proteins that contribute to the inhibition of the error-prone MHEJ include Ku and histone H1. Treatment of extracts with flap endonuclease 1 antiserum significantly reduced MHEJ. Addition of a 17-bp intervening sequence between the microhomologous sequences significantly reduced the efficiency of MHEJ. Thus, this cell-free assay provides a platform for evaluating factors modulating end joining.

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.

Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1.The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFP fusion gene. The expression of this reporter gene can be assessed with the 124I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (124I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped 124I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of 124I-FIAU was also compared with that of an exogenous hypoxic cell marker, 18F-fluoromisonidazole (FMISO).Our results showed that 124I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of 124I-FIAU and 18F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between 124I-FIAU and 18F-FMISO.This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable ( p un ) allele results in reversion to the wildtype pink-eyed dilution ( p ) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo.

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54-/-Ku80-/- mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54-/-Ku80-/- mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54-/-Ku80-/- animals accumulated foci of γ-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Purpose: To develop a novel super-resolution time-resolved 4DMRI technique to evaluate multi-breath, irregular and complex organ motion without respiratory surrogate for radiotherapy planning. Methods: The super-resolution time-resolved (TR) 4DMRI approach combines a series of low-resolution 3D cine MRI images acquired during free breathing (FB) with a high-resolution breath-hold (BH) 3DMRI via deformable image registration (DIR). Five volunteers participated in the study under an IRB-approved protocol. The 3D cine images with voxel size of 5×5×5 mm3 at two volumes per second (2Hz) were acquired coronally using a T1 fast field echo sequence, half-scan (0.8) acceleration, and SENSE (3) parallel imaging. Phase-encoding was set in the lateral direction to minimize motion artifacts. The BH image with voxel size of 2×2×2 mm3 was acquired using the same sequence within 10 seconds. A demons-based DIR program was employed to produce super-resolution 2Hz 4DMRI. Registration quality was visually assessed using difference images between TR 4DMRI and 3D cine and quantitatively assessed using average voxel correlation. The fidelity of the 3D cine images was assessed using a gel phantom and a 1D motion platform by comparing mobile and static images. Results: Owing to voxel intensity similarity using the same MRI scanning sequence, accurate DIR between FB and BH images is achieved. The voxel correlations between 3D cine and TR 4DMRI are greater than 0.92 in all cases and the difference images illustrate minimal residual error with little systematic patterns. The 3D cine images of the mobile gel phantom preserve object geometry with minimal scanning artifacts. Conclusion: The super-resolution time-resolved 4DMRI technique has been achieved via DIR, providing a potential solution for multi-breath motion assessment. Accurate DIR mapping has been achieved to map high-resolution BH images to low-resolution FB images, producing 2Hz volumetric high-resolution 4DMRI. Further validation and improvement are still required prior to clinical applications. This study is in part supported by the NIH (U54CA137788/U54CA132378).

MRI has excellent soft tissue contrast and can provide both anatomical and physiological information. It is becoming increasingly important in radiation therapy for treatment planning, image-guided radiation therapy, and treatment assessment. It is critically important at this time point to educate and update our medical physicists about MRI to prepare for the upcoming surge of MRI applications in radiation therapy. This session will review important basics of MR physics, pulse sequence designs, and current radiotherapy application, as well as showcase exciting new developments in MRI that can be potentially useful in radiation therapy. Learning Objectives: 1.To learn basics of MR physics and understand the differences between various pulse sequences 2.To review current applications of MRI in radiation therapy.To discuss recent MRI advances for future MRI guided radiation therapy Partly supported by NIH (1R21CA165384).; W. Miller, Research supported in part by Siemens Healthcare; G. Li, My clinical research is in part supported by NIH U54CA137788. I have a collaborative research project with Philips Healthcare.; J. Cai, jing cai

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

The ability to repair DNA double-strand breaks is essential to maintain chromosomal stability. Virtually all soft tissue sarcomas contain chromosomal instabilities, including clonal aberrations and cytogenetic aberrations. However, the relevance of DNA-dependent protein kinase (DNA-PK) in the pathogenesis of soft tissue sarcoma has not been clarified. The main aim of this work is to compare the prognostic impact of genotypic imbalance in low-grade soft tissue sarcomas of the extremities, and to correlate this with the translational level of DNA-PK. This study investigated 28 adult low-grade malignant spindle cell tumours of the extremities, predominantly fibrosarcomas, for loss of heterozygosity (LOH) and microsatellite mutation on flanking regions of each DNA-PK subunit, with identical immunophenotypes. Twelve different polymorphic markers flanking the specific loci of three subunits comprise the genetic map of DNA-PK, at 22q13, 2q35, and 8q11. Translational activity was also analysed by western blot and conventional immunohistochemistry. The overall sarcoma 5-year survival rate was 61.7%. LOH was identified in the specific coding region of DNA-PK in 39.29% for the DNA-PK catalytic subunit (cs), 17.86% for Ku70, and only 7.14% for Ku80. A positive LOH for DNA-PKcs was shown to be a significant factor for poor survival (log rank test p = 0.0160). Immunoreactivity and immunoblot results correlated with the loss of DNA-PKcs allotype in soft tissue sarcoma (Fisher's exact test p = 0.0037). Ku70 and DNA-PKcs were almost identical in terms of immunoreactivity. In conclusion, whereas microsatellite mutation seems an uncommon event during the evolution of low-grade fibrosarcoma of the extremities in adults, the loss of DNA-PKcs defines a biologically more aggressive subset.

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout ( DNA-PKcs −/−) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs −/− mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm −/− mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK cs -deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK cs gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

We present evidence that inactivation of the Ku70 gene leads to a propensity for malignant transformation both in vitro and in vivo. In vitro, Ku70 −/− mouse fibroblasts displayed an increased rate of sister chromatid exchange and a high frequency of spontaneous neoplastic transformation. In vivo, Ku70 −/− mice, known to be defective in B but not T lymphocyte maturation, developed thymic and disseminated T cell lymphomas at a mean age of 6 months with CD4 + CD8 + tumor cells. These findings directly demonstrate that Ku70 deficiency facilitates neoplastic growth and suggest a novel role of the Ku70 locus in tumor suppression.

The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK). We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation. Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression. We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element. This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter. This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles. Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation. Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 ( Ku70 −/− ). Like Ku80 −/− mice, Ku70 −/− mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80 −/− mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4 + CD8 − and CD4 − CD8 + T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

Previously, we found that extremely low frequency (ELF) electric fields were able to elicit an approximate 3.5-fold increase in heat shock gene expression, a response which may have applicability to cancer therapy. Based on recent studies demonstrating the ability of magnetic fields to influence gene expression, we hypothesized that low level static magnetic fields may be able to affect heat shock gene expression while avoiding some of the clinical difficulties that arise with electric fields. Transfected rat primary cells in monolayer were exposed to magnetic fields of 1 to 440 mT for 16, 24, or 48 h starting at 24 and 48 h post transfection. Heat shock protein (HSP70) expression, as indicated by a promoter linked luciferase reporter, was followed for up to 96 h and showed a dependence on flux density, exposure duration, and start time post transfection. A nonlinear response was observed for increasing flux density with a maximum of a 3.5-fold increase in expression for 48 h of exposure starting 48 h after transfection. These results demonstrate an enhancement of gene expression similar in magnitude to that observed with external electric field exposure, while eliminating many of the clinical complications.

Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression.

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20-60 min at 43 degrees C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFkappaB p65 subunit, suggesting perturbation of NFkappaB activation. A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFkappaB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock. Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1. These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1. The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.

Purpose: To optimize T2-based respiratory-correlated 4DMRI for motion simulation, we evaluated the image quality after 4D reconstruction based on the two respiratory surrogates acquired independently and concurrently during the same breathing period. Methods: Both internal navigator and external bellows were employed simultaneously during a 4DMRI acquisition on seven volunteers scanned coronally and sagittally under an IRB-approved protocol. The navigator was positioned at the right diaphragm dome while the bellows was placed at the upper abdomen. Using the navigator as the primary surrogate, prospective amplitude-binning was used to fill respiratory bins and anatomic slices to complete the amplitud-elocation table on the fly to reconstruct a single-breath 4DMRI. Five bins in both inhalation and exhalation were used, forming a 10-bin 4DMRI. Following the same binning method, a bellows-based 4DMRI was then reconstructed retrospectively using a MATLAB programs developed for the rebinning and data analysis. The two surrogates were compared on 1D breathing waveforms and 4D image quality around the diaphragms. The waveform comparison included correlations in inhalation and exhalation, hysteresis analysis by ellipsoidal fitting, and histogram analysis for slice redistribution in bellows-based bins. Image quality comparison between the navigator-based and bellows-based 4DMRI included motion artifact visualization and phase-shift quantification at the right and left diaphragms. Results: The correlation between the navigator and bellows is usually higher in inhalation than exhalation. Their non-linear relationship is represented using ellipsoidal fitting with minor-to-major-axis ratio of 0.2–0.5 and by cross-correlation with phase shifts (0.2–2). Overall, navigator-based 4DMRI has superior image quality, although artifacts at anatomy distant to the navigator, such as the left diaphragm, are observed. Conclusion: This is the first time that two-differently-binned 4DMRI image sets from the same breathing period have been directly compared. Navigator-based 4DMRI is generally superior to bellows-based 4DMRI, making it clearly suitable for tumor delineation in future clinical applications. This study is in part supported by the NIH (U54CA137788/U54CA132378). Also, this research is collaborated with Philips Healthcare.

Purpose: To develop a novel approach to improve cervical spine (c-spine) curvature reproducibility for head and neck (HN) patients using optical surface imaging (OSI) with two regions of interests (ROIs). Methods: The OSI-guided, two-step setup procedure requires two ROIs: ROI-1 of the shoulders and ROI-2 of the face. The neck can be stretched or squeezed in superior-inferior (SI) direction using a specially-designed sliding head support. We hypothesize that when these two ROIs are aligned, the c-spine should fall into a naturally reproducible position under same setup conditions. An anthropomorphous phantom test was performed to examine neck pitch angles comparing with the calculated angles. Three volunteers participated in the experiments, which start with conventional HN setup using skin markers and room lasers. An OSI image and lateral photo-picture were acquired as the references. In each of the three replicate tests, conventional setup was first applied after volunteers got on the couch. ROI-1 was aligned by moving the body, followed by ROI-2 alignment via adjusting head position and orientation under real-time OSI guidance. A final static OSI image and lateral picture were taken to evaluate both anterior and posterior surface alignments. Three degrees of freedom can be adjusted if an open-face mask was applied, including head SI shift using the sliding head support and pitch-and-roll rotations using a commercial couch extension. Surface alignment was analyzed comparing with conventional setup. Results: The neck pitch angle measured by OSI is consistent with the calculated (0.2±0.6°). Volunteer study illustrated improved c-spine setup reproducibility using OSI comparing with conventional setup. ROI alignments with 2mm/1° tolerance are achieved within 3 minutes. Identical knee support is important to achieve ROI-1 pitch alignment. Conclusion: The feasibility of this novel approach has been demonstrated for c-spine curvature setup reproducibility. Further evaluation is necessary with bony alignment variation in patient studies. This study is in part supported by the NIH (U54CA137788).

The role of mammalian 70-kDa heat shock protein (hsp70) in regulating cellular response to heat shock was examined by using three closely related rat cells: control Rat-1 cells, thermotolerant Rat-1 (TT Rat-1) cells, and heat-resistant M21 cells, a derivative of Rat-1 cells that constitutively overexpress human hsp70. In all these cells, after a prescribed heat shock, the level of the phosphorylated form of heat shock transcription factor HSF1 and that of HSF1 capable of binding to its cognitive DNA sequence heat shock element (HSE) exhibit similar time dependence. The amount of a constitutive HSE-binding activity (CHBA), on the other hand, inversely correlates with those of the two aforementioned forms of HSF1. The recovery kinetics from heat shock are different for the three cell lines, with the thermal-resistant TT Rat-1 and M21 cells showing faster recovery in terms of the state of phosphorylation of HSF1 and its ability to bind HSE or in terms of the reappearance of CHBA. Treatment with okadaic acid, a serine/threonine phosphatase inhibitor, delays the recovery kinetics of Rat-1 cells but not that of thermal-resistant M21 cells. These results are interpreted in terms of a role for hsp70 in the recovery of heat-shocked mammalian cells.