Takehiko Kakizaki

LET-Dependent Survival of Irradiated Normal Human Fibroblasts and Their Descendents

Abstract Hamada, N., Funayama, T., Wada, S., Sakashita, T., Kakizaki, T., Ni, M. and Kobayashi, Y. LET-Dependent Survival of Irradiated Normal Human Fibroblasts and Their Descendents. Radiat. Res. 166, 24–30 (2006). Evidence has accumulated showing that ionizing radiations persistently perturb genomic stability and induce delayed reproductive death in the progeny of survivors; however, the linear energy transfer (LET) dependence of these inductions has not been fully characterized. We have investigated the cell killing effectiveness of γ rays (0.2 keV/μm) and six different beams of heavy-ion particles with LETs ranging from 16.2 to 1610 keV/μm in normal human fibroblasts. First, irradiated confluent density-inhibited cultures were plated for primary colony formation, revealing that the relative biological effectiveness (RBE) based on the primary 10% survival dose peaked at 108 keV/μm and that the inactivation cross section increased proportionally up to 437 keV/μm. Second, cells harvested from primary colonies were plated for secondary colony formation, showing that delayed reproductive death occurred in a dose-dependent fashion. While the RBE based on the secondary 80% survival dose peaked at 108 keV/μm, very little difference in LET was observed in the RBE based on secondary survival at the primary 10% survival dose. Our present results indicate that delayed reproductive death arising only during secondary colony formation is independent of LET and is more likely to be dependent on initial damages having been fixed during primary colony formation.

Heavy‐ion‐induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication

The aim of the present study was to clarify the mechanisms of cell death induced by heavy‐ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy‐ions. In this experiment, the limited number of cells (0.0001–0.002%, 5–25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8–14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy‐ions was induced mainly by direct cell‐to‐cell communication, such as GJIC, which might play important roles in bystander responses. (Cancer Sci 2009; 100: 684–688)

Cell cycle arrest and apoptosis in Caenorhabditis elegans germline cells following heavy-ion microbeam irradiation

Purpose: To investigate positional effects of radiation with an energetic heavy-ion microbeam on germline cells using an experimental model metazoan Caenorhabditis elegans. Materials and methods: The germline cells were irradiated with raster-scanned broad beam or collimated microbeam of 220 MeV 12C5+ particles delivered from the azimuthally varying field (AVF) cyclotron, and subsequently observed for cell cycle arrest and apoptosis. Results: Whole-body irradiation with the broad beam at the L4 larval stage arrested germ cell proliferation. When the tip region of the gonad arm was irradiated locally with the microbeam at the L4 stage, the same arrest was observed. When the microbeams were used to irradiate the pachytene region of the gonad arm, at a young gravid stage, radiation-induced apoptosis occurred in the gonad. In contrast, arrest and apoptosis were not induced in the non-irradiated neighboring region or the opposite gonad. Similar results were confirmed in the c-abl-1 (mammalian ortholog of cellular counterpart of Abelson murine leukemia virus) mutant that is hypersensitive to radiation-induced apoptosis. Conclusion: These results indicate that the microbeam irradiation is useful in characterizing tissue-specific, local biological response to radiation in organisms. DNA damage-induced cell cycle arrest and apoptosis were observed in locally irradiated regions, but there was little, if any, ‘bystander effect’ in the nematode.

Role of DNA-PKcs in the bystander effect after low- or high-LET irradiation

Purpose: To investigate the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the medium-mediated bystander effect for chromosomal aberrations induced by low-linear energy transfer (LET) X-rays and high-LET heavy ions in normal human fibroblast cells. Materials and methods: The recipient cells were treated for 12 h with conditioned medium, which was harvested from donor cells at 24 h after exposure to 10 Gy of soft X-rays (5 keV/µm) and 20Ne ions (437 keV/µm), followed by analyses of chromosome aberrations in recipient cells with premature chromosome condensation methods. To examine the role of DNA-PKcs and nitric oxide (NO), cells were treated with its inhibitor LY294002 (LY) and its scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), respectively. Results: Increased frequency of chromosome aberrations in recipient cells treated with conditioned medium from irradiated but not from un-irradiated donor cells was observed which was independent of radiation type. Bystander induction of chromosome aberrations in recipient cells was mitigated when donor cells were treated with LY before irradiation and with c-PTIO after irradiation, and was enhanced when recipient cells were treated with LY before treatment of recipient cells with conditioned medium from irradiated donor cells. Conclusion: Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to un-irradiated bystander cells, leading to the induction of bystander chromosome aberrations partially repairable by DNA-PKcs-mediated DNA damage repair machinery, such as non-homologous end-joining repair pathways.

Brown adipose tissue in cetacean blubber.

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.

Dependence of the bystander effect for micronucleus formation on dose of heavy-ion radiation in normal human fibroblasts

Ionising radiation-induced bystander effects are well recognised, but its dependence on dose or linear energy transfer (LET) is still a matter of debate. To test this, 49 sites in confluent cultures of AG01522D normal human fibroblasts were targeted with microbeams of carbon (103 keV µm(-1)), neon (375 keV µm(-1)) and argon ions (1260 keV µm(-1)) and evaluated for the bystander-induced formation of micronucleus that is a kind of a chromosome aberration. Targeted exposure to neon and argon ions significantly increased the micronucleus frequency in bystander cells to the similar extent irrespective of the particle numbers per site of 1-6. In contrast, the bystander micronucleus frequency increased with increasing the number of carbon-ion particles in a range between 1 and 3 particles per site and was similar in a range between 3 and 8 particles per site. These results suggest that the bystander effect of heavy ions for micronucleus formation depends on dose.

ANALYSIS OF THE LETHAL EFFECTS OF LOW-DOSE RADIATION ON GLIOMA CELLS: RELATIONSHIP OF THE BYSTANDER EFFECT AND METAL ELEMENTS

Investigation of the radiation-induced bystander effect plays an important role in the understanding of the mechanisms of radiation response after low-dose irradiation. Sphingomyelinase (SMase) was activated by radiation and required the metal element for its activation. For further elucidation of the bystander effect, we investigated the relationship between its induction by acid SMase and a factor secreted from the irradiated tumor cells. In the cell culture medium transfer experiment after irradiation at a dose of 0.1 Gy, cell death was induced in non-irradiated cells. However, when cells received prior treatment with SMase inhibitor, cell death was not induced. When fluctuations in the activation of SMase and metal elements were detected, both intracellularly and extracellularly after irradiation, an increase in SMase activity and Zn concentration occurred within the cells at 5 min and outside of the cells at 15 min after irradiation. This increase in zinc concentration at 15 min after irradiation was suppressed by treatment with SMase inhibitor. These results suggest that activation of SMase, which is related to the bystander effect, is dependent on zinc.

Whole-Body Counter Evaluation of Internal Radioactive Cesium in Dogs and Cats Exposed to the Fukushima Nuclear Disaster.

As a result of the 2011 nuclear incident that occurred at the Fukushima Nuclear Power Plant, a large number of abandoned dogs and cats were left within the disaster zone. A small number of these animals were rescued and cared for at shelters. Prior to the dispersal of these animals to their owners or fosterers, we evaluated the degree of internal radiocesium contamination using a specially designed whole-body counter. We conducted 863 non-invasive measurements of gamma rays due to internal radioactive cesium for 68 dogs and 120 cats at one shelter. After plotting graphs of 137Cs density we generated exponential functions of decay from seven dogs and six cats. From the regression formulae, we were able to determine the biological half-lives as 38.2 days for dogs and 30.8 days for cats. We found that in dogs there was a correlation between the biological half-life of radioactive cesium and age. Using our data, we estimated whole-body densities for each cat and dog at the time when they were rescued. We found that there were deviations in the data distributions among the different species, likely due to the timing of rescue, or living habits prior to rescue. A significant correlation was found when extracted feline reproductive organs were analyzed; the coefficients for the estimation of whole-body densities were approximately 7-fold higher than those based on the extracted feline reproductive organs. This may be due to the fact that majority of the radioactive cesium accumulates within muscular tissue with less distribution in other organs. It is possible to plan the appropriate management period in an animal shelter based on the use of the biological half-life of radioactive cesium calculated in this study. We believe that the correlations we uncovered in this work would be of great use for the management of companion animals in the event of a future nuclear accident.