口丸 高弘

The microenvironment of solid tumors is characterized by low pO(2) that is well below physiological levels. Intratumoral hypoxia is a major factor contributing to cancer progression and is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells near blood vessels, poor lymphatic drainage resulting in high interstitial pressure, and irregular blood supply through immature tumor vasculature. Hypoxia-inducible factor-1 (HIF-1) is the main transcription factor that regulates cellular responses to hypoxia. Cellular changes induced by HIF-1 are extremely important targets for cancer therapy. Therefore, targeting strategies to counteract HIF-1-active cells are essential for cancer therapy. In this study, we introduce a novel strategy for targeting HIF-1-active cells.

A cell chip was developed for the examination of biological damage of cells irradiated by high-energy alpha particles. A CR-39 track detector was employed as a chip substrate to identify high-energy charged particles traversing target cells. Moreover, the patterning of a photopolymer layer spatially controlled the cellular adhesiveness on the chip substrate. HeLa cancer cells were cultured on a micropatterned photopolymer layer. In this way, all the cells on the chip were individually addressed through the block number in the photopolymer pattern. The biocompatibility of the cell chip was examined through a viability test with fluophor reagent and measurement of the cell proliferation rate. HeLa cancer cells on the chip were irradiated with alpha particles and stained with a fluorescent probe molecule for DNA damage detection. The CR-39 substrate was etched by means of an alkali solution during cell incubation. The HeLa cells and alpha tracks were successfully observed by microscopy at once. It was confirmed that fluorescent spots corresponding to DNA damage were located in the direction of the major axis of oval alpha tracks. © Atomic Energy Society of Japan.

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIF alpha) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIF alpha protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIF alpha and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases.

X-ray microbeam transport in radiophotoluminescent (RPL) glass was examined by fluorescence microscopy. A silver-activated phosphate glass dosimeter plate was irradiated with an X-ray microbeam emitted from a 50 kV micro focus X-ray tube equipped with a glass capillary. A RPL region (wavelength ~650 nm) was observed with UV light excitation along the X-ray microbeam track in the glass dosimeter plate. The divergence of the RPL track was small owing to the X-ray energy dependence of the divergence of the microbeam, which was determined from the critical angle of the X-ray reflection on the inner wall of the glass capillary. The image of the RPL track was analyzed using photon transport and light trace simulation codes. It was confirmed by comparison between the RPL observation and simulation results that the RPL image in the silver-activated phosphate glass accurately projected the energy-deposited track along the X-ray microbeam. © 2009 The Japan Society of Applied Physics.

To identify individual cells exposed to a X-ray microbeam in a cell population, we developed a biocompatible microchamber-array chip using UV lithography of photopolymer SU-8. The center-to-center distance between microchambers is 50 μm including a wall of 15 μm height. Using the microchamber-array chip, we performed tracking of individual exposed cells. Sample cells loaded in a microchamber array were selectively irradiated with the X-ray microbeam under microscopic observation. All the irradiated cells were indexed by the array arrangement of the microchambers. For about 24 h of post-irradiation incubation, the irradiated cells were identified successfully by time-lapse observation. In addition, the induction of radiation effects was observed in identified cells using immunofluorescence. © 2008 The Author(s).

Three-dimensional (3D) image processing for analysis of track etching in a Solid State Nuclear Track Detector (SSNTD) was performed. A SSNTD in contact with boron nitride films was exposed to epithermal and thermal neutrons. In chemical etching process, sequential images of etch-pits on the surface of the SSNTD were obtained with a developed observation system. By use of a digital-image-processing technique, 3D images were reconstructed from data on sequential surface images of the track detector for particles released due to 10 B (n, α)7 Li reactions. © 2008 Elsevier Ltd. All rights reserved.

A commercially available radiophotoluminescent (RPL) glass dosimeter was used for the profile measurement of an X-ray microbeam. The X-ray microbeam of 10 μ m diameter was produced by a microfocus X-ray tube and a glass capillary. An RPL glass plate of 1 mm thickness was irradiated with the X-ray microbeam. After the irradiation, the RPL image for the X-ray microbeam on the glass plate was observed with a conventional fluorescence microscope. In the RPL image analysis, the RPL photons around 600 nm wavelength were detected by exposure to UV photons in the wavelength range from 330 to 380 nm. The profile of the X-ray microbeam was well measured with the RPL glass dosimeter and the RPL intensity was proportional to the X-ray fluence. Normal human fibroblasts cells, AG01522B, were grown on the RPL glass plate for single cell irradiation. A pre-selected living cell was irradiated by the X-ray microbeam and the absorbed dose was estimated to be about 3 Gy. After the irradiation, the targeting cell was stained with anti-γ-H2AX antibody, Alexa Fluor® 488 and propidium iodide, to visualize the effects on DNA double strand breaks in the nucleus of the cell. In fluorescence microscopy, foci in the nucleus corresponding to the phosphorylation of serine 139 of H2AX and the X-ray beam spot were simultaneously observed at the position of the targeting cell. © 2007 Elsevier Ltd. All rights reserved.

An in-situ observation system was developed to record time-lapse images of etch pits formed on the surface of a solid-state nuclear track detector (SSNTD), CR-39. The CR-39 track detector was irradiated with alpha and Li particles produced by a neutron capture reaction, 10B(n,α)7Li. The time-lapse images of the etched surface of the detector were carefully observed to elucidate the etch pit formation process. Pit-evolution images were constructed by digital image processing of the data of the time-lapse images. In addition, a finite element model of SSNTD was used to simulate the etch pit formation. Pit-evolution image analysis and computational simulation were performed to reveal the etch pit formation obtained via the incidence angle of energetic particles on a CR-39. © 2008 The Japan Society of Applied Physics.

A tabletop micro X-ray beam irradiation system has been developed to study radiation effects on living cells. The microbeam system is composed of a micro focus X-ray tube, a capillary for X-ray guide, an X-ray semiconductor detector for fluorescent X-ray analysis, and an inverted microscope. A beam profile measurement was performed and the size of the focused beam was 10 μm in diameter [full-width at half-maximum (FWHM)] with a divergence of 5.1 mrad. The data on the microbeam measurement were approximately in agreement with a simulation of X-ray optical traces for the capillary. In addition, the microdosimetric characterization for single cell irradiation was performed with the beam profile measurement and a photon-electron transport simulation. The maximum of the dose rate for the sample cells set in the system was estimated to be 0.05 Gy/s. In a preliminary experiment, single yeast cells were irradiated with the X-ray microbeams, and the data on the survival rate of the cell samples for the X-ray doses were obtained. Then, the microbeam irradiation system is useful to investigate the radiation effects on the living cells. © 2006 IEEE.

A tabletop micro X-ray beam irradiation system has been developed to research radiation effects on living cells. The microbeam system is composed of a micro focus X-ray tube, a capillary for X-ray guide, an X-ray semiconductor detector for fluorescent X-ray analysis and an inverted microscope. A beam profile measurement was performed and the size of the focused beam was. 10 pm in diameter (FWHM) with the divergence of 5.1 mrad. The data on the microbeam measurement was approximately agreement with a simulation of X-ray optical traces for the designed capillary. In addition, the microdosimetric characterization for the single cell irradiation was performed with the beam profile measurement and a photon-electron transport simulation. The maximum of the dose rate for the sample cells set in the system was estimated to be 0.05 Gy/s. In a preliminary experiment, single yeast cell was irradiated with the X-ray microbeam, and the data on the survival rate of the cell samples for the X-ray doses was obtained. Then, the microbeam irradiation system is useful to research the radiation effects to the living cells.

A. tabletop micro X-ray beam irradiation system has been developed to research radiation effects on living cells. The microbeam system is composed of a micro focus X-ray tube, a capillary for X-ray guide, an X-ray semiconductor detector for fluorescent X-ray analysis and an inverted microscope. A beam profile measurement was performed and the size of the focused beam was 10 μm in diameter (FWHM) with the divergence of 5.1 mrad. The data on the microbeam measurement was approximately agreement with a simulation of X-ray optical traces for the designed capillary. In addition, the microdosimetric characterization for the single cell irradiation was performed with the beam profile measurement and a photon-electron transport simulation. The maximum of the dose rate for the sample cells set in the system was estimated to be 0.05 Gy/s. In a preliminary experiment, single yeast cell was irradiated with the X-ray microbeam, and the data on the survival rate of the cell samples for the X-ray doses was obtained. Then, the microbeam irradiation system is useful to research the radiation effects to the living cells. © 2005 IEEE.