Human leukemic MOLT-4 cells undergo apoptosis after X-irradiation through p53-dependent pathway. p53 (wild type) is stabilized and increased after X-irradiation. To examine this process more in detail, we here measure the p53 level in a single cell by flow cytometry. Irradiated cells were fixed and permeabilized, stained with anti-p53 mAb, and analyzed on FACSCalibur (Becton Dickinson). The cellular p53 protein level was increased by 4-5 h after the irradiation, and then decreased gradually, when cells were exposed to 0.05-9.1 Gy: at D10-dose or less, the level was recovered to the level of unirradiated cells and cells were survived; whereas at the dose higher than D10-dose, the cells rapidly developed apoptosis, and the level decreased gradually and finally reached to the level lower than that of control cells. These results suggest that in recovering cells, the cellular p53 level returned to the normal level by physiological proteolysis systems, whereas in dying cells, the level decreased to the level lower than the normal level by the leakage from the cell.

View full abstract

Nitroxide Tempo is known to act as an anti-ROS agent and an inhibitor of ROS-induced apoptosis. Nonetheless, we observed sensitization of hyperthermia-induced apoptosis by 5 mM Tempo in U937 cells. We investigated the sensitization mechanisms. 5 mM Tempo or 44C/10 min heating was non-apoptotic to U937 and PC-12 cells. However, the combined treatment markedly sensitized U937 and PC-12 cells to apoptosis, as 44C/30 min induced. Although levels of Bcl-2, Bcl-XL and Bax in lysates were not altered, Bax was rapidly activated and translocated to mitochondria after the combined treatment and 44C/30 min, followed by cytochrome c release. High levels of [Ca²⁺]_i and [Ca²⁺]_m sustained long after the combined treatment and 44C/30 min. As a result, the mitochondrial membrane potential was decreased by mitochondrial Ca²⁺ overload-mediated permeability transition. Generations of mitochondrial superoxide and ATP were also decreased after such treatments. The combined treatment and 44C/30 min activated JNK 1, but the inhibitors of JNK activation had no effect on enhanced apoptosis of U937 cells, but a partial inhibition in PC-12 cells. Our results suggest that sensitization of Tempo/heat-induced apoptosis may involve at least two mechanisms: (1) increased Bax-mediated OMM permeability and (2) opening of Ca²⁺-mediated permeability transition pore. Both mechanisms result in cytochrome c release, mitochondrial dysfunction, and cell death.

View full abstract

We have previously reported that the low-dose UVB irradiation (~0.1J/cm²) or treatment with benzene metabolite, hydroquinone (HQ) suppressed apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis plays an important role in tumor progression and its malignancy and in the promoting resistance to cancer therapy. To clarify the antiapoptotic mechanism, we tried the comprehensive detection of the apoptosis regulation factors in the low-dose UVB-irradiated or HQ-treated NIH3T3 cells using two-dimensional (2-D) difference gel electrophoresis (Ettan^TM DIGE). Of about 4000 protein spots detected, 42 spots and 45 spots were found to differ significantly in survived cells due to the UVB irradiation and HQ-treatment, respectively, in comparison with apoptosis-undergoing cells. In the selected spots, 32spots in UV-irradiated cells and 36 spots in HQ-treated cells were identified using MALDI-TOF/ TOF-MS. The detection of apoptosis regulation molecules in the process of apoptosis inhibition by the low-dose UVB irradiation or HQ-treatment using DIGE system would lead to develop a better base of knowledge for tumor induction and for designing new anti-tumor drugs in the future.

View full abstract

Since UV generally causes a rapid cell death and ionizing radiation induces a delayed form of apoptosis, the activation of signal transduction by radiation would be different between γ-rays and UV. In order to identify the signaling regulators associated with apoptotic cell death, we analyzed cellular proteins responding to γ or UV irradiation using two dimensional gel electrophoresis (2DE). In this study, Jurkat cells were irradiated with 15 Gy of γ-rays or 20 J/m² of UV, both of which show a similar effect on cell killing, incubated for various time intervals, and then treated with digitonin for preparing their cytosolic extracts. Comparison of the high resolution 2DE protein patterns of the extracts from irradiated and un-irradiated cells showed differences in many spots including protein modifications. These protein spots were analyzed after tryptic digestion by peptide mass fingerprinting using a MALDI-TOF/TOF mass spectrometry. Interestingly, three different spots were identified as acidic ribosomal protein P2, one of the ribosomal stalk components, and found that the amount of dephosphorylated form of this component increased with increasing time of incubation after γ and UV irradiation. Further 2DE analyses of proteins responding to radiation are in progress.

View full abstract

To clarify the mechanism by which the radon hot spring prevents cancer or not, in this study, blood was collected from the residents in the Misasa hot spring district and in the control district, and the level of a representative cancer-suppressive gene, p53, and the activity of a representative antioxidant enzyme, superoxide dismutase (SOD), were analyzed as indices. As a result, the level of serum p53 protein in the males in the Misasa hot spring district was 2-fold higher than that in the control district, with a significant difference. In the females in the Misasa hot spring district, SOD activity was approximately 15 % higher with statistical significance than that in the control district, and exceeded the reference range of SOD activity despite advanced age. These results suggested that routine exposure to radon at a concentration about 3 times higher than the national mean by the residents in the Misasa hot spring district induces trace active oxygen in vivo, potentiating products of cancer-suppressive gene and antioxidant function. As the p53 protein level was high in the residents in the Misasa hot spring district, apoptosis of cancer cells may readily occur. These factors may have led to the low cancer-related mortality rate.

View full abstract

Transcriptional activity of p53-target genes is enhanced by low dose ionizing radiation. However the detailed mechanisms for promoter activation by radiation are not elucidated. One of the reasons is that a reporter vector responsive to radiation has not been developed. Recently plenty of evidences were reported indicating that association of p53 with its recognition sequence of DNA is dependent on the chromatin structure. For instance, it was revealed, by in vitro experiments, that native p53 binds with higher affinity to the chromatin-assembled DNA than to double-stranded oligonucleotides. In vivo DNase I-hypersensitive site analyses showed that p53 recognition sites in the p21, 14-3-3&sigma, and KARP-1 genes are resistant to nuclease digestion even after gene induction by irradiation. We have constructed a reporter vector containing the luciferase gene under the control of the p21 gene promoter by using adeno-associated virus, with which the transgene can be integrated into chromosomes. When the reporter construct was transduced into MCF-7 cells, a remarkably high radiation-response was observed. It was revealed that luciferase expression was significantly induced by 0.5 Gy of X rays. This result indicates that the mechanism for activation of p21 gene promoter by low-dose radiation can be analyzed by use of this vector.

View full abstract

A total of 4000 SPF B6C3F1 mice were divided into four groups [three irradiated groups and one control group], each consisting of 500 male and 500 female mice. Irradiation was performed using ¹³⁷Cs gamma-rays at dose-rates of 20, 1 and 0.05 mGy/day with accumulated doses equivalent to 8000, 400 and 20 mGy, respectively.
Pathological examination showed that the most frequently observed lethal neoplasms were malignant lymphomas, liver, lung, and soft tissue neoplasms in males and, malignant lymphomas and soft tissue neoplasms in females. Deaths due to these neoplasms occurred at an earlier age in both sexes irradiated with 20 mGy/day group. Non-lethal neoplasms of the Harderian glands were frequently observed in the 20 mGy/day group in both sexes. In females, non-lethal neoplasms of the lung and endocrine organs were frequently observed in 20 mGy/day group. (The work is supported by grants from Aomori Prefecture, Japan.)

View full abstract

To elucidate the mechanism of the radon therapy, we had started the basic study on the internal movement of inhaled radon. In this study, gamma rays from the lung, heart, and brain of mice, which inhaled radon with a radon atmosphere, were measured. A mineral, which is used in an artificial radon hot spring, was put in a cage and sealed over one month. The concentration of radon in the cage was measured by PicoRad liquid scintillation vial comparing with that in the hot bath room with a high concentration of radon at Misasa Hospital Center of Okayama University Medical School. The concentration of radon in the cage was estimated to be 6000 Bq/m³. Mice were put in the cage for 50 min. Their hearts, lungs, and brains were enucleated and measured by a high-purity germanium detector. Gamma rays of radon progeny were detected from the hearts and lungs of mice but the brains. It is suggested that the inhaled radon could reach the heart but the brain in this experimental condition.

View full abstract

The catalase activities in blood and organs of the acatalasemic (C3H/AnLCsb-Csb) mouse of C3H strain are lower than those of the normal (C3H/AnLCsaCsa) mouse. We examined the effects of post low-dose (0.5 Gy) X-ray irradiation, which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in the acatalasemic or normal mice. In the results, the 0.5 Gy irradiation after carbon tetrachloride administration decreased the glutamic oxaloacetic and glutamic pyruvic transaminase activity in the acatalasemic mouse blood to a level similar to that of the carbon tetrachloride no treatment acatalasemic mouse blood, in contrast to high-dose (15 Gy) irradiation. In the same manner, pathological showed that carbon tetrachloride-induced hepatopathy was inhibited by 0.5 Gy irradiation. These findings suggest that low-dose irradiation after administration accelerated the rate of recovery from hepatopathy induced by carbon tetrachloride, in contrast to high-dose irradiation.

View full abstract

An exposure condition of tritium radiation from nuclear fusion reactor could be a long-term exposure with low dose rate. The biological effects of low dose (rate) radiation are not clear because any suitable detection system has not been established. Regarding to mutation induction by high LET radiation such as neutrons, the reversed dose rate effect has been reported when the dose rate is lower than a certain value. This might be caused by hypersensitivity of G2/M cells for mutation induction by high LET radiation. However, it is not clear whether this phenomenon could be seen in the case of tritium radiation. To examine the low dose rate effect of tritium, we established a hypersensitive mutation detection system using hamster cells carrying a human X-chromosome. We have tested mutation induction by tritiated water at dose rate between 0.15 and 4.4 cGy/h. Our results suggest that mutation frequency seems to be slightly increased at lower dose rate tritium radiation but the increase level was much less than that by neutrons.

View full abstract

An adaptive response at whole body level has been reported in terms of survival rate and the clonogenisity of bone marrow cells on the spleen of irradiated mice. Conventionally, pre-irradiation was carried out at high dose-rate; here, we examined, by measuring endogenous spleen colonies, whether the adaptive response was induced with low dose-rate irradiation.
Whole body irradiation of C57BL/6N female mice (6 week-old) was carried out with ¹³⁷Cs gamma rays (1.2mGy/hr). On the day 40, 50 or 60, the low dose-rate irradiated and control mice were exposed to 6Gy of X-rays (260kV, 3.3mA, 0.5mm Al + 0.3mm Cu filter, 0.46Gy/min). On the 12 day after the X-irradiation, the spleen was removed and fixed in Bouin's solution, and then the number of colonies on the surface of the spleen was counted.
No difference was found in the number of the colonies in the mice pre-irradiated for 40 or 50 days and the control (6Gy only) group. On the other hand, the number of spleen colonies was increased in mice pre-irradiated for 60 days. These results showed that the low dose-rate pre-irradiation induced the adaptive response.

View full abstract

Our previous study suggested the possibility that the lifespan shortening induced in B6C3F1 mice by low dose rate (20 mGy/day) gamma-ray irradiation could be caused by early occurrence of malignant lymphoma. The loss of heterozygosities (LOHs) were investigated on chromosomes 4, 11, and 12 by the simple sequence length polymorphism (SSLP)-based PCR assay in 60 lymphoma samples. LOHs were frequently found at a region of D12Mit133 on chromosome 12 in radiation-induced lymphomas (15.6%), and control ones (42.9%). The result was different from that found in radiation-induced thymic lymphomas reported, while possible candidate genes should be investigated. This study was carried out by financial support from Aomori Prefecture, Japan.

View full abstract

Our previous studies about the effects of continuous low dose rate gamma-irradiation on lifespan and carcinogenesis showed a plausible correlation between the life-shortening and an early occurrence of some neoplasms in mice irradiated with 20 mGy/day; however, the molecular mechanism underlying the early occurrence after irradiation remains to be elucidated. In the present study, we analyzed chromosomal aberrations in malignant lymphomas, which were the most frequently observed, lethal neoplasm in both the non-irradiated control and the irradiated groups, by array-based comparative genomic hybridization (array-CGH) method. We constructed a mouse BAC microarray that contained 672 DNA fragments covering the following important genetic loci: oncogenes, tumor suppressor genes and candidate disease genes identified near the common retrovirus integration sites listed in Retroviral Tagged Cancer Gene Database. The present analysis in a total of 21 lymphomas showed that deletion in a part of chromosomes 19 was observed in about 40% and 18% in non-irradiated control and irradiated groups, respectively. In addition, amplification in a distal part of X chromosome and trisomy 15 were associated preferentially with non-irradiated control and irradiated groups. This work was carried out with financial support from Aomori Prefecture, Japan.

View full abstract

We have been investigating effects of low-dose-rate radiation (LDR) on immortal human cells established by introducing the hTERT gene. GI-phase-arrested AT and normal cells were irradiated with LDR (0.3mGy/min) continuously for a maximum of 1 week or high-dose-rate radiation (HDR; 2Gy/min). In normal cells, the frequency of HPRT deficient mutations after LDR is much lower than that after HDR. Results of multiplex PCR analysis indicated that both the size and frequency of deletions in mutants induced in normal cells by LDR are also smaller than with HDR. In contrast, in AT cells, the mutation frequency after LDR is only a little lower than that after HDR. In addition, frequency of deletions seemed similar for LDR and HDR. These results suggest that effect of LDR radiation may appear more severely in cells defective in DNA repair, like AT cells.

View full abstract

Female C57BL/6N mice (5 week-old, 35-36 in each group) were irradiated with ¹³⁷Cs gamma rays at 0.35, 0.70 or 1.2 mGy/hr for 34 days. The mice were then injected (s.c) with 0.05 mg of methylcholanthrene (MC) dissolved in olive oil and the irradiation was continued. The levels of oxidative DNA damage product, 8-OhdG, in urine of all of mice were measured every month. The cumulative tumor incidence after 220 days following the MC injection in non-irradiated control group was 23 %. There was a delay in the incidence of tumor in the irradiated groups. The cumulative tumor incidence was also suppressive in the irradiated groups: 22 % in 0.35-mGy/hr group, 11 % in 0.70-mGy/hr, and 6 % in 1.2-mGy/hr. No difference was formed in the level of 8-OhdG between the non-irradiated control group and the irradiated groups, or among the irradiated groups. These results suggested that the inhibitory effects on the tumor incidence by the low-dose rate irradiation were due to certain mechanism offer than the suppression of endogenous oxidative damage.

View full abstract

Although there have been piled up many evidences showing the beneficial effects of low dose/ low dose rate radiation, precise mechanisms for the effects are still unclear. We have been reported that continuous exposure on low dose rate gamma-ray ameliorates diabetes and elongates lifespan in db/db, a mouse model for type II diabetes caused by genetic defect of leptin receptors. Here we report the results of pathological and physiological analysis for the ameliorating effect of low dose rate radiation.
Db/db mice were continuously exposed to gamma ray with dose rates between 0,01-1 mGy/h. Blood glucose of db/db mice was three times higher than control litter mates, db/+ mice, and gradually increased as aging. Blood insulin of db/db was also much higher than control littermates, and it gradually decreased as aging. Continuous irradiation suppressed the decrease in blood insulin without marked effect on the hyperglycemia. Pathological analysis demonstrated hyperplasia of pancreatic Langerhans islands in irradiated db/db mice. These results suggest that continuous exposure on low dose rate gamma-ray ameliorates diabetes by maintaining secretion of insulin from pancreatic Langerhans islands.

View full abstract

We reported the suppressive effect of low dose-rate irradiation on tumorigenesis in methylcholanthrene (MC)-injected mice. To elucidate the mechanism underlying the tumor suppression we examined the tumor cell rejection, using a TD50 (tumor dose 50) assay, in irradiated mice. Female C57BL/6N mice were irradiated with ¹³⁷Cs γ-rays at 0.4, 0.7, or 1.2 mGy/hr for 1-8 weeks. Then appropriate numbers of tumor cells prepared from a MC-induced tumor were injected into groins and axillae of mice to estimate TD50 value. Mice received a total dose of 250 mGy, regardless of dose rate, showed maximum TD50 value (3-5 fold compared to the non-irradiated controls). The increase in TD50 was not observed at total dose of 50, 150, 400, 750, or 1200 mGy. These results indicated that the ability of tumor cell rejection was increased at a certain condition and that the low dose-rate irradiation never decreased the tumor rejection even at as much as 1200mGy.

View full abstract

We examined mutations induced by ionizing radiation with or without radioadaptive response at the Hprt locus in mouse m5S cells. We analyzed the Hprt gene in 6-thioguanine-resistant mutants by amplification of all the nine exons of the mouse Hprt gene. Our results showed that the proportion of partial deletions was much higher in mutants isolated from the pre-exposed radioadaptive culture than in non-primed irradiated culture. Although about half of the mutants that retained the whole Hprt gene represented point mutations, others showed normal genomic sequences. We detected no Hprt mRNA in those mutants that have the normal Hprt gene, indicating that suppression of gene expression plays an important role in radiation-induced mutagenesis.

View full abstract

To understand a mechanism of radio-adaptive response induced by a low-dose radiation, whole-body irradiation studies using laboratory animals have a significant implication. We previously showed that pre-irradiation with 0.5 Gy of X-rays induces radio-resistance in C57BL/6L mice by suppressing bone marrow death induced by irradiation with 6 Gy without a recovery of total number of peripheral blood cells. In the present study, we examined a possible new role of bone marrow stem cells in radio-adaptive mice by bone marrow transplant experiments. To acquire radio-adaptive condition, the mice were maintained for two weeks after exposure to 0.5 Gy of X-rays, and then sacrificed to harvest bone marrow stem cells. The recipient mice were exposed to acute irradiation with 6.25-7.0 Gy, and then received a bone marrow transplant. After 12 days, a number of spleen colonies in the transplanted mice were scored. The result indicated that pre-irradiation with 0.5 Gy reduced spleen colonies. In some 7.0 Gy-irradiated recipient mice, bone marrow death observed in spite of the appearance of a few spleen colonies, was suggesting that only function of hematopoiesis is not enough to overcome bone marrow death. Those serial studies explain a piece of mechanism of radio-adaptive response, suggest that bone marrow stem cells play a new role to avoid bone marrow death rather than hematopoieses.

View full abstract

We already reported that low dose-rate radiation induced the gene expression of certain p53 related genes weakly but significantly. In this report, we elucidated that p53 would contribute significantly to molecular response to low dose-rate irradiation as well as to high dose-rate, and found the difference in patterns of p21^waf1 gene expression between high and low dose-rate. Method: Human acute lymphoblastic leukemia cell line MOLT-4 and B3 cells, a stable clone derived from MOLT-4 transfected with murine mutant p53, were irradiated with low dose-rate γ-rays at a dose rate of 1.1 mGy/hr. As high dose-rate radiation, we used X-rays at a dose rate of 0.25 Gy/min (150 kV, 5mA, filter: 1.0 mm Al + 0.2 mm Cu). Gene expression was measured by real time reverse transcriptase PCR method. Result: 1) In MOLT-4 cells, the level of p21^waf1 gene expression increased up to 1.5-fold by the low dose-rate irradiation, and the level was kept at least for 311 days. 2) In B3 cells in which the function of endogenous p53 was inhibited through a dominant-negative effect, the induction was suppressed. 3) After the low dose-rate irradiation, the level of gene expression decayed more slowly than the case for the irradiation with 0.08 Gy at the high dose-rate which induced p21^waf1 to similar extent as the low dose-rate irradiation.

View full abstract

The biological effects of low-LET ionizing radiation are characterized by dose-rate effects (DREs). It is thought that the frequency of chromosome aberration or mutation induced by low dose-rate decreases rather than high dose-rate. However, recent data show that DREs on chromosome aberration have not been observed in mice receiving the X-rays at dose rate of less than 55 mGy/day. This result indicates that DNA damages induced by very low dose rate may remain unrepaired. The generation and repair of radiation-induced DNA damages in cells have been recently examined by enumeration of phosphorylated histone H2AX (γ-H2AX) foci. Using published data on γ-H2AX foci in this study, we focus here that the number of γ-H2AX foci per cells becomes constant at 24 hours after X-rays irradiation at dose of less than a few ten mGy. We hypothesize that DNA damages induced by X-rays with below a few ten mGy repairs until the same amount of DNA damages at 24 hours after acute irradiation. Consequently this suggests that DRE would not occur among the mice receiving the X-rays irradiation at dose rate of less than a few ten mGy/day. γ-H2AX foci formation may be useful to elucidate the mechanism of DRE.

View full abstract

Prognosis after irradiation is closely related to the radiation sensitivity of stem cells in several tissues, such as the small intestine and bone marrow. Although the biological significance of radiosensitivity has been recognized, it has been quite difficult to analyze its mechanism in stem cells owing to the difficulty of their identification and isolation. We irradiated E14 cells, a mouse embryonic stem cell line, with 0, 0.5, 5 and 10 Gy (0.98Gy/min) of ionizing radiation and harvested the irradiated cells one hour later. Total RNA fractions prepared from the cells were used for HiCEP analysis and approximately 23,000 transcripts were detected. 75 transcripts were induced and more than 600 suppressed by irradiation at 5 Gy, and 50 transcripts were induced and 212 were suppressed at 0.5Gy. 13 transcripts were induced only by 0.5 Gy irradiation, suggesting that these are low dose-specific genes. Now we are focusing our analysis on the low-dose range from 0.05 Gy to 0.5 Gy, and isolating and sequencing the induced genes.

View full abstract

Germ-line mutation induced by ionizing radiation is suspected as a cause for the increased genetic risk, including the cancer risk, in successive generations. Two conflicting results, the negative one on the first-generation progeny of the atomic bomb survivors at Hiroshima and Nagasaki and the positive one among the human population in the Belarus area after the Chernobyl accident, have been reported for the effect of ionizing radiation on germline mutation in human populations. The mouse specific locus test has been successfully applied to evaluate the genetic effects of radiation by other researchers. However, the frequencies of spontaneous mutation have varied in different series of experiments, suggesting that the number of mice used in the test was not sufficient for a statistical analysis. Therefore, we started to establish a new experimental system to detect germline mutation at the molecular level by the direct DNA sequencing of genomic DNA using the automated DNA sequencer. We identified several mutations in the offspring derived from the male mice that were exposed with 3 Gy of gamma ray. On the other hand, any mutational events have not been identified in the offspring derived from un-irradiated parents. We are currently investigating these mutations in detail, and the results will be presented.

View full abstract

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme which is transcriptionally activated by various stimuli. We investigated the effect of caffeic acid derivatives and ferulic acid derivatives on HO-1 gene mRNA in a mouse monocyte-macrophage cell line, RAW 264.7 and compared with that of cysteamine. When cells were incubated with CAPE, a remarkable increase of HO-1 mRNA was observed. The mRNA of HO-1 reached the peak 4 hrs after addition of CAPE, and then declined 2 hrs later. Cysteamine also activated the transcription of HO-1 gene. But a much higher concentration of cysteamine was necessary than CAPE, and the maximal induction by cysteamine was much smaller. Caffeic acid ethyl ester and two ferulic acid derivatives, curcumin and ethyl ferulate, also enhanced mRNA of HO-1 gene at the same concentration with CAPE. To examine the cell specificity of the activation human hepatocarcinoma cell line, HepG2, and mouse macrophage cell line, J774.1, were treated with CAPE. As a result mRNA for HO-1 increased in J774.1, but not in HepG2. Thus the activation of HO-1 by CAPE is specific to macrophage and it is supposed that the activation mechanism by CAPE is different from that by cysteamine.

View full abstract

We reported previously that adaptive response to low dose irradiation decreased with aging. In order to investigate its mechanisms, we searched the variant expressions of proteins caused by high or low dose irradiation in cells from variously aged rats. Proteomics using 2D-PAGE and peptide mass fingerprinting is suitable for the study on both the expressions and the post-translational modifications of proteins. Alterations in the relative intensity of protein spots appearing on proteome profiles were analyzed using PDQuest software (Bio-Rad) and identifications of protein spots were performed using MALDI-TOF MS analysis. When glial cells cultured from young rats were irradiated at the dose of 5 Gy, the relative abundance of 2 spots increased on 2D gels, as compared with control gels. These spots corresponded to peroxiredoxin II and latexin. While in cells from aged rats, both peroxiredoxin II and latexin were not increased significantly by 5 Gy-irradiation. In the present work, we also studied the protein spots phosphorylated by irradiation, and will discuss the effects of aging on cell responses to radiation in the presentation.

View full abstract

Owing to accomplishment of the human genome project and development of molecular biological techniques including microarray analysis, it become possible to investigate the common feature of radiation-responsive genes and genes involved in radiation sensitivity of mammalian cells. Marko, et al. have identified human genes responsive either to beta-rays or gamma-rays by a microarray analysis. Based on their data we attempted to reveal a common feature of the radiation responsive genes by examining the structure of the 5'UTR or the presence/absence of TATA element. It has been established that the 5'UTR plays a role in regulation of translational efficiency of the gene by the following mechanisms; 1) a cap-dependent scanning, and 2) internal ribosome entry by internal ribosome entry segment (IRES). It has been shown that IRES is often utilized in translation of genes controlling cell growth/death, apoptosis-related genes and protooncogenes, and that under stress condition, translation by IRES is more preferred compared to a cap-dependent scanning. We found that the majority of radiation-responsive genes contain a long GC-rich 5'UTR, and suggested preference of IRES-mediated translation. On the other hand, in regard to the structure of the promoter region, we notified that a substantial number of radiation-responsive genes contains Sp1 recognition sequences instead of the TATA element.

View full abstract

One of the mechanisms of radiation-induced reoxygenation is a decrease in oxygen consumption of irradiated tumor tissues. An in vitro experiments, however, shows rather an increase in cellular oxygen consumption rate after photon irradiation. No report has been made for post-irradiation oxygen consumption after high LET radiation. In the present study, we measured the oxygen consumption rate in mouse lymphomas after photon and carbon-ion irradiation. The cellular oxygen consumption rate in both L5178Y and M10 cells (a radiosensitive mutant of L5178Y) increased shortly after both 5-40 Gy of X rays and 2-20 Gy of 80 keV/μm carbon ions irradiation, and then returned to the value before irradiation. The time of occurrence of peak oxygen consumption for L5178Y and M10 was 12 and 24 hours, respectively, after irradiation, and was independent of radiation quality and dose. The peak oxygen consumption in M10 was 200-300% of controls, and was higher than that in L5178Y.

View full abstract

According to former study of radiation biological physics, direct exposure to ionizing radiation induces DNA damages and chromosomal aberrations in cells received direct traverse of radiations and does not affect to other cells. However, this understanding has been challenged by recent studies in which effects are observed on cells received no radiation exposure but are in the neighborhood of irradiated cells. It is reported as a radiation induced bystander effects, which transmits radiation induced effects to surrounding cells received no radiation exposure. GM14511, human lymphoblast cells were attached on dishes as samples. Samples were partly masked in order to distinguish irradiated and unirradiated part. Using heavy ion accelerator at TIARA of JAERI-Takasaki, we exposed the whole or part of masked samples to 20Ne (LET=430 keV/μm) ion beam. After irradiation, cells were incubated for 0-12 hours and then aberrations on chromosomes of irradiated and nonirradiated samples were observed by using premature chromosome condensation method. Result of observation demonstrated that number of cells with damages on chromosomes were larger than the number of cells received radiation exposure. Furthermore, we distinguished cells with degree of their chromosomal aberration, heavily damaged and lightly damaged ones. The kinetics of those two kinds of aberration indicated the radiation induced bystander effect on chromosomal aberration.

View full abstract

Gut injury is critical for carbon-ion radiotherapy to visceral organs. We have investigated effects of fractionated irradiation with carbon ions on gut crypt survivals. We here investigated and reported that bFGF, a cytokine, plays an important role in determining radiosensitivity after low-LET carbon-ion irradiation. C3H female mice received repeated irradiation of 1 Gy/fraction with an interval of 4 hr. Jejunum was removed after final irradiation, and served for immunostaining with anti-bFGF antibody and western blotting. In some experiments, gut received topup doses 2 hr after an IV injection of anti-bFGF antibody that was carried out 2hr after a final 1-Gy irradiation. Gut was removed 3.5 days after the topup irradiation, and removed to be fixed in formalin for H.E. Staining. Results were (1) bFGF was detected for guts received 3 repeated doses or over, (2) bFGF gradually decreased with time, but was still detectable 60 hr after the final irradiation, (3) administration of anti-bFGF antibody was not effective against mice that received 3 repeated doses, but reduced survival levels in mice received 11 repeated doses. We conclude that bFGF are responsible to the radioresistance induced by low-LET carbon ions.

View full abstract

We investigated resistance relationship between tumor sensitivity and DNA precursor incorporation after irradiations under mice breathing air with or without tumor blood clamping. We transplanted HSG cells to mice right leg. When HSG grew at 7-8mm diameter, the mouse leg was clamped to shut down blood flow, and then irradiated by 15Gy gamma ray or carbon ions (290MeV /u; LET: 74/ micrometer). The clamping changed tumor sensitivity so that tumor grew faster than non-clamped tumors. However, in the measurement after the irradiation 24hours, the tendency of ¹⁴C-thymidine uptake increase was seen in the tumor which made hypoxic condition. This indicates that ¹⁴C-thymidine uptake is a good measure of not only therapeutic efficiency but also radioresistance induced by hypoxia.

View full abstract

By subjecting human colon tumor HCT116 cells to a repeated X-ray irradiation, radiosensitive variants were obtained. The colonies appeared after X-ray exposure (8 Gy) were pooled at two weeks, grown for a week, and subjected to a subsequent X-ray exposure. After 5th exposure, twenty colonies were isolated and their radiosensitivity was examined. Radiosensitivity of the cells that have experienced a repeated X-ray exposure (once to five times) did not differ from that of parental HCT116 cells when examined as a mixed cell population. There was, however, a significant variation in radiosensitivity among the cells derived from the 20 individual colonies. Such a variation may not be due to the heterogeneity in parental HCT116 cells, because the colonies derived from non-irradiated reference did not exhibit such a variation. The cells derived from four colonies were significantly radiosensitive: their survival rates at 6 Gy were 5-10-fold lower than that of parental cells. The analysis of transcriptional response by quantitative PCR method using the Light Cycler apparatus revealed that the increase of MDM2 transcripts after 6-Gy exposure was slightly decreased in the four radiosensitive clones compared to parental cells or other clones, which may related the their enhanced radiosensitivity.

View full abstract

The non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase in colon cancer cells, and induce NSAID activated gene (NAG-1), which is a divergent member of TGF-β super family. It has reported that NAG-1 is induced by 10Gy irradiation, and is downstream of p53 signaling pathway. To clarify the effects of irradiation on colon cancer cells, we analyzed the frequencies of apoptosis of HCT116 (human colon cancer cell line) after lower dose irradiation, and evaluated the relationship with the expressions of NAG-1 and p53. HCT116 (p53 wild), p53 knock-out HCT116 (p53KO) and HCT15 (p53 mutant) were irradiated with 3Gy γ-rays, and then harvested up to 24h after irradiation. The frequencies of apoptosis increased at 6h, and then decreased 24h after irradiation in p53 wild cells. However, in p53KO cells and p53 mutant cells, there were no remarkable changes. In p53 wild cells, the expressions of p53 and NAG-1 mRNA reached peak level 6 and 9h after irradiation, respectively. Also, the expressions of p53 protein showed biphasic increase 3 and 9h after irradiation, the expressions of NAG-1 protein gradually increased in time dependent manner. On the other hand, there were no remarkable expressions of NAG-1 of protein in p53KO cells and p53 mutant cells. We suggested that the expression of NAG-1 induced apoptosis in p53 dependent manner after irradiation.

View full abstract

The production characteristics of ⁹C radioactive beams in the secondary beam line at HIMAC have been investigated and the optimal conditions to produce a ⁹C beam with higher production rate and purity were found. Physical and radiobiological experiments have been carried out for the beam with low intensity (approximately 10⁴pps or 0.3-0.5 Gy/h). Radiobiological effects on HSG cell survival at different depths, i.e. different LET along the penetration of the beam were obtained. Taking gamma-rays with simulated low dose rate as the reference, higher RBE for the beam at the depths around its Bragg peak (around 80 keV/um) than ¹²C beam have been revealed. In following studies, a spot scanning system was used to deliver the ions to cell samples so as to increase the dose rate, and comparison cell experiment using ¹²C beam will be done in almost the same condition as the ⁹C beam (low dose rate, large energy spread, etc).

View full abstract

During the non homologous end joining (NHEJ) pathway of DNA double strand break (DSB) repair, it is known that DNA-PKcs, the catalytic subunit of DNA-PK, phosphorylates itself and this autophosphorylation is thought to be very important in the process of NHEJ. We have used a phosphor-specific DNA-PKcs antibody to study the effect of radiation in human cells. The foci formation of the phosphorylated DNA-PKcs induced by x-rays and high LET carbon ion beams were investigated in normal human fibroblast cells (HFLIII) and Ligase IV deficient cells (180BR). The number of foci of the phosphorylated DNA-PKcs was significantly higher in 180 BR cells than that of HFLIII cells even without irradiation. After x-irradiation (2 Gy), the peak of phosphorylated DNA-PKcs focus formation was 1hour in both cell lines. Although the number of foci were gradually reduced and returned to almost the levels of unirradiated conditions after 24 hours incubation. After high LET carbon ion beams (2 Gy), the peak of focus formation was significantly delayed in both cell lines when compared with x-irradiated cells. This delay was very distinct in irradiated 180BR cells. These results indicate that high LET radiation seems to disturb the machinery of NHEJ and the study with heavy ion particles may help further clarify the mechanism behind the NHEJ process.

View full abstract

Clinical trials indicate that the RBE of high LET radiation is large for slow-growing tumors. We here used 11 transplantable tumors with different growth time for each, and compared RBE values of 290 MeV/u carbon ions (74 keV/micrometer). NFSa fibrosarcoma is spontaneously arisen in C3H mice and other 10 radiation-induced tumors were transplanted in hind legs, and irradiated with either Cs-137 gamma rays or 290 MeV/u carbon ions. Tumor growth delay time and specific growth delay, (i.e., tumor growth time for experimental group) / (tumor growth time for un-irradiated group), were calculated from tumor growth time (i.e., days for a tumor to reach 5 times initial volume), and used to obtain isoeffect doses. TG time for un-irradiated control ranged from 3 days to 9.6 days. RBE of carbon ions ranged from 2.1 to 3.4. It is concluded that tumor growth rate is less important than anticipated for a RBE determinant of carbon-ion radiation.

View full abstract

To investigate the mutagenicity of the bystander effect in BNCT, we mixed the electroporated-BSH cells that accumulated ¹⁰B inside cells, and no-boron cells. These cells were irradiated by neutron (KUR: 5MW) and the mutagenicity was measured by the mutant frequency in the HPRT locus. The molecular structure of HPRT mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. The mutant frequency was increased by the bystander effect compared with control study. Molecular structure analysis of mutants induced bystander effect indicated that point mutation were dominant and the incidences of total and partial deletions were decreased compared with original mutants induced by neutron irradiation. These results suggested that the bystander effect in BNCT induced mutants in different mechanism with original neutron damage.

View full abstract

We studied the localization of phosphorylated H2AX (γ-H2AX) in cultured human fibroblasts after irradiation with heavy ion beams. Asynchronous human normal fibroblasts (NB1RGB) were irradiated with X-rays, carbon ion beam (LET is about 88 keV/μm), Si ion beam (220 keV/μm) and Fe ion beam (440 keV/μm) at room temperature. γ-H2AX in irradiated cells was measured from 0 to 24 h after irradiation by using flow cytometry. γ-H2AX increased just after irradiation of each radiation and reached maximum around 30 min and then decreased. Therefore, Foci of γ-H2AX on cell nucli were observed under the laser scanning confocal microscopy at 30 min after irradiation. Number of foci per nucleus was increased with increasing dose for each beam.

View full abstract

Carbon-ion beam, accelerated by HIMAC in the National Institute of Radiological Sciences (NIRS), is a candidate for use in therapy of malignant melanoma on eyes. The purpose of this study is to estimate the relative biological effectiveness (RBE) in the wide dose range using monolayer cells and multicellular spheroids. We used one human malignant melanoma cell line, HMV-I. Surviving fractions of cells exposed to X-rays and carbon-ions at a low dose region (0 - 8 Gy) and a higher region (8 - 15 Gy) were obtained using monolayer cells and multicellular spheroids, respectively. The linear-quadratic (LQ) model fits well in survival data of monolayer cultures after both X-rays and carbon-ions at lower dose region. The multi-target single-hit (H) model fits well in those of spheroids at higher dose region. In addition, we could fit with a formula (LH) combined both the models, as well as the multi process model, to survival data in wide dose range, when both monolayer and spheroid data was combined. We could estimate RBE values of carbon-ions from low to very high dose region by converting the surviving fractions of spheroids into monolayer cells surviving fractions with the LH combined fit equation.

View full abstract

Accelerated heavy ions have been known to induce complex clustered DNA damages leading to nonrepairable DNA double-strand breaks (DSBs), which predominantly cause cell death and chromosomal aberrations. However it has not yet cleared the molecular mechanisms, especially, whether DSB repair proteins could recognize heavy ion induced DSBs. Here we report the molecular response of DSB repair proteins on the DSBs induced by high LET iron ions. Irradiation of accelerated iron ions ((⁵⁶Fe, 90 MeV/u) was performed at the RIKEN ring cyclotron facility. An X-ray machine operating at 250 kVp was used as reference irradiation. The RBE of 1000 keV/μm iron ions for HeLa cells was 1.16. The induction of TUNEL positive clustered DSBs were observed immediately after iron ion irradiation. NBS1 and γ-H2AX, which were essential for homologous recombination (HR), formed highly localized foci, which could not be observed after 5 Gy of X-irradiation. Large NBS1 foci also colocalized with γ-H2AX and were remained even 16 h after irradiation. In addition, the prolonged G2 arrest was detected after iron-ion irradiation in contrast to after X-irradiation. These results suggest that the DSBs induced high LET heavy-ion cannot be efficiently repaired by HR, and then the repair foci cannot be dissolvable and prolonged G2 arrest.

View full abstract

Radiation impairs some functions of the central nervous system, which is one of the radiation-resistant tissues in the body. It is known that these radiation-induced alterations are dependent on the quality of radiation, but the underlying mechanisms are still unclear. In this study, we investigated the effects of high- linear energy transfer (LET) heavy-ions and low-LET X-ray on the release of glutamate, the major excitatory neurotransmitter in the central nervous system, in the hypothalamus of rats measured by in vivo brain microdialysis. Total body and head, but not abdominal, heavy-ion (carbon) irradiation induced a significant increase in glutamate levels to approximately 150% of the basal level at 1.5h of the irradiation, and the release gradually increased during the observation period. However, total body or abdominal, but not head, X-ray irradiation caused a significant increase in glutamate release to approximately 150 - 170% of the basal level within 1h of the irradiation. The increased release returned to the baseline in the following 1h. These results indicated that heavy-ion irradiation directly increases the release of glutamate in hypothalamus, while peripheral pathway such as vagal afferents may be predominantly involved in the X-ray-induced glutamate release.

View full abstract

Radioprotectors clinically available are almost organic compounds. Progress in discovering biological functions of inorganic compounds provides an abundance of pharmaceutical candidates. Na₃VO₄, one of the vanadate compounds, showing potential biofunctions including antiapoptosis, was tested for possible radioprotective effect. Pregnant ICR mice were X-irradiated with 0.5 to 4.0 Gy on E 11 or E12. Na₃VO₄ dissolution was intraperitoneally injected 2 hours later at a dose of 5 or 15 mg/kg. Half of the dams were sacrificed on E18 and the fetuses were examined. Reminders were left for delivery and the neonates were monitored for life, death, gross malformation and a 30-day survival. Administration of Na₃VO₄ alone on E11 at 15 mg/kg increased significantly fetal killing and malformation, however no toxicity was observed in other groups. When administrated at 15 mg/kg, the detrimental effects of fetal killing and prenatal malformation from 3.5 Gy were significantly reduced. Postnatal death was also markedly suppressed in 1.0 Gy group. Efficacy of Na₃VO₄ at 15 mg/kg and its combination with other apoptosis inhibitors are under investigation in 0.5 Gy group. Na₃VO₄ could protect mouse fetal development from radiation detrimental effect in late organogenesis. Exploration and development of new inorganic compounds for clinical application as radioprotectors would have great possibilities in the future.

View full abstract

Purpose: When the radiotherapy is applied in the malignant tumor in genital organs, the normal digestive organ receives the irradiation simultaneously. The digestive organs are easily caused the radiation injury because the radiosensitivity is high. Melatonin has a radical-scavenging action, and little side effect. In present study, we studied on the protective effect of melatonin against radiation-induced digestive injury in mice intestine. Methods: Six-week-old Slc:ICR male mice and fourteen-week-old C3H/He female mice were used. Mice were given whole-body irradiation with a ¹³⁷Cs gamma ray and LET 50 keV/µm carbon ion. The mice were orally administered 1 ml of either the 1% carboxymethyl cellulose sodium salt or melatonin. The mice were sacrificed at 3.5 days after the exposure. The jejunum was removed, fixed in formalin and then stained with haematoxylin and eosin. The number of crypts per transverse circumference was counted microscopically for 10 histlogical sections of each mouse. Result: Melatonin protected small intestine from the injury induced by gamma ray. The protective effect was dependent on the plasma melatonin concentrations. The intestine was protected at the highest extent 0.5 h after the administration. For carbon ions, however, melatonin didn't. Conclusion: Melatonin may be a potent protector against the intestinal damage as a side effect in radiotherapy. However, further experimental and clinical studies on this subject are need.

View full abstract

We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collect and irradiated in vitro. In this study, human whole blood was in vitro exposed to ¹³⁷Cs γ rays or 50 keV/µm carbon ions in the presence or absence glycine betaine (one of the beer components). Glycine betaine reduced the chromosome aberrations (dicentric) caused by either γ rays or carbon ions of 4 Gy. The maximum protection for γ rays by glycine betaine was 37%, while the maximum protection for carbon-ion by glycine betaine was 20%. C3H/He female mice, aged 14 weeks, received an i.p. injection of glycine betaine at 15 min before whole-body irradiation with γ rays or 50 keV/µm carbon ions. Glycine betaine i.p. injection significantly (p<0.05) increased the percent survival for γ rays and carbon ions. Oral administration of glycine betaine was not effect. It is concluded that glycine betaine is a potent protector against damage caused by radiation with direct and indirect actions.

View full abstract

Purpose: Tetrahydrobiopterin (BH₄) is a cofactor of aromatic amino acid (phenylalanine, tyrosine, tryptophan) hydroxylases and of nitric oxide synthase (NOS). It is reported that BH₄ eliminates superoxide anion radical and inhibits NO-induced apoptosis. The effect of BH₄ on radiation-induced micronucleus frequency of RAW264.7 cells was investigated in this study. Methods: Mouse macrophage-like RAW264.7 cells were irradiated with 5Gy γ-ray in the presence or absence of sepiapterin (SP), a precursor of BH₄ biosynthesis. To determine micronucleus frequency, cells were fixed to a microscope slide and stained with Giemsa and micronuclei were scored under microscopy. Cellular glutathione level and antioxidant enzyme activity were assayed according to the conventional methods. Results and Discussion: Micronucleus frequency resulting from 5Gy γ-rays given at 18 and 24h after treatment of 1µM SP significantly decreased compared to micronuclei induced by γ-ray irradiation alone. The cellular glutathione level and antioxidant enzyme activity at 18h after treatment of 1µM SP did not show any significant changes compared to those of the non-treated cells. The pretreatment of N-acetyl-5-hydroxytryptamine (NAS), an inhibitor of BH₄ biosynthesis, blocked the reduction of γ-ray induced micronuclei by SP. These results suggest that the reduction of γ-ray induced micronuclei by SP would be due to BH₄ which is biosynthesized in the cells.

View full abstract

Low LET radiation generates toxic free radicals and the radical-induced injury to nulear DNA makes serious damages in living cells. Although endogenous mechanisms to diminish toxicity of the free radicals are studied in the cell-free system, their function in the living cells are not clarified. To reveal the initial damages and the protection mechanism, we established the method to quantitate radiation damage and the radioprotection in the living cells. When murnie macrophage-like cell line RAW264.7 was used, x-ray-induced nuclear damage could be measured reporducibly with limited deviation. The frequencies of both micronucleated and growth-inhibited cells were increased by x-ray of doses from 0.2 to 3.0 Gy at dose-dependent manner. Among various antioxidative compunds, thiols showed the most protective effect on both the micronucleation and growth-inhibition. It was suggested the protection levels were dependent upon not only the membrane permeability of the molecule but also the effect to modify the intracellular thiol levels. When constitutive exprssion gene for glutathion peroxidase with modification in amino acid sequence were stably introduced into the cells, radiation-induced growth inhibition was decreased. These results show contribution of the regulation of intracellular thiols to the nuclear damage by ionizing radiation.

View full abstract

We investigated the effect of edaravone on the radiation in mouse intestine.
C3H mice were irradiated to the whole body at the total dose of 18 Gy using X-rays (150 kVp, 5 mA, 1.0 mmAl+ 0.2 mmCu filter, 0.30 Gy/min). Mice were administered intraperitoneally with edaravone 1 hour before irradiation. At 4 hour after irradiation, jejunum of mice was removed and stained with TUNEL. The number of apoptosis per crypt was measured. There was difference between edaravone-treated group and the control group in the number of apoptosis per crypt.
The number of apoptosis per crypt of edaravone-treated mice was significantly lower than that of the control group. The result indicated that the premedication of edaravone protected from the intestinal injury by apoptosis during the radiation.

View full abstract

Maitake mushroom extract called MD-fraction prepared from the fruit body of Maitake (Grifola frondosa) was reported to have no harmful side effects to normal tissues, increase immune-competent cell activities and indicate anti-tumor activities. In the present study, the radiation protective effect of MD-fraction of Maitake mushroom (Tanabe Co.,LTD) were examined. Two groups of male ICR mice, 10-20 weeks old, were orally administered MD-fraction for seven consecutive days, and the control group received saline, before whole-body exposure to gamma radiation (4 and 8Gy). After irradiation, measurements of body weight and hematocrit value and the estimation of DNA damage in tissue and blood cell by the comet assay were conducted. The oral administration of MD-fraction before exposure to gamma ray irradiation was apt to be effective in decreasing the DNA damage in bone marrow cells and blood cells. The administration of MD-fraction also tended to prevent the depilation due to irradiation.

View full abstract

Purpose: It has been reported that high dose of nitric oxide (NO), which is a high reactive radical, exerts cytotoxic effects, leading to DNA damage, micronucleus (MN) formation, and mutation. Though, low dose of NO may have an opposite effect and protect cell from the death caused by irradiation or ROS stimuli, the mechanism underlying these responses remains unclear. Effect of NO on the radiation-induced micronucleus frequency of RAW264.7 calls was investigated in this study. Methods: Mouse macrophage-like RAW264.7 cells were irradiated with 5Gy gamma-ray in the presence or absence of sodium nitroprusside (SNP), NOC5, NOR4, or S-nitrosoglutathione (GSNO), a chemical NO donor. To determine micronuclei frequency, cells were fixed to a microscope slide and stained with GIMSA and micronuclei were scored under microscopy. Cell cycle was analyzed by flow cytometry. The cellular GSH level was investigated by using conventional method. Results: Micronucleus frequency resulting from 5Gy gamma-rays given at 3~12h after treatment of 0.25mM SNP significantly decreased compared to the number of the value induced by 5Gy gamma ray irradiation alone. Same effect was observed in other NO donors. Conclusions: These results suggest that NO reduces micronucleus frequency induced by 5Gy gamma ray irradiation in RAW264.7 cells.

View full abstract

In this study, we used the beta-1,3-D glucan configuration which we extracted than Saccharomyces cerevisiae (macro-glucan) and measured immunocompetence activity, an anti-tumor effect, a radiation protection effect. Experimental animal used C3H mouse (male, seven weeks) and did dosage of macro-glucan of 200 mg/kg to 800 mg/kg. After the dosage, we measured immunocompetence activity by measuring white blood cells, lymphocytes, NK, LAK cell activity. In addition, we measured an anti-tumor effect of macro-glucan. Transient increase was noted in white blood cells after the macro-glucan dosage, lymphocytes change. In addition, by cell damage activity determination, as for NK, the LAK cell activity, 400, 800 mg/kg administrated group did activity in significance in comparison with control group. In an experiment of an anti-tumor effect, tumor growth restraint was observed in administrated group. We think that an anti-tumor effect was noted by immunocompetence activity including NK cell activity of statement above. A macrophage participates in immunocompetence activity such as the above and a factor of an anti-tumor effect and this lets you discharge all kinds of site Cain and amplifies immune function, and I think that we attack a cancer cell. We think that infectious disease depression after bombardments with immunocompetence activation by the macro-glucan dosage and anti-oxidation action. Immunocompetence activity of macro-glucan, an anti-tumor effect and a radiation protection effect were confirmed in this study.

View full abstract

(Purpose) Hyperthermia was performed at over 42.5C in clinicaly, but we find that below 42.5C as 41C still has effects of enhanced the anti-tumor effect of the drug and increase of the immunological activity. (Materials and Methods) SCC-VII tumor cells were transplanted subcutaneously in the thigh of C3H male mice 6 weeks old. For heat treatment of the tumor, a thermostatic circulating water bath(41C and 42C,30min) was used on ten mice in each group which were control group, drug alone group, heat alone group and drug plus heat group. Also we measured number of the white blood cells and lymphocyte cells, and activate of immunological activity as NK cells activity using Cr-51 labeled EL-4 cells assay. Also we measured the drug uptake in the tumor using HPLC units. (Results and Discussion) Mild-hyperthermia has effect of enhance the anti-tumor effect by increase of drug uptake due to the increase of blood flow in the tumor when combined with drug and mild-hyperthermia. The number of white blood cells and lymphocyte cells and NK cells activity as immunological activity were increased after hyperthermia. These results suggest that mild-hyperthermia can reduce the side effects due to the reduction of the drug doses and increase of immunological activity can maintain the QOL levels for patients.

View full abstract