Long-lived radicals (LLRs), assigned as sulfinyl radicals (SLF: R-S-O•), are produced when mammalian cells are γ-irradiated at room temperature. Addition of Vit. C to the cells AFTER irradiation causes the reduction of mutation frequency levels simultaneously with the decrease in the concentration of LLRs. On the carbon ion radiation to mammalian cells followed by addition of Vit. C or Ribose-Cysteine(RibCys), levels of mutation frequency are also decreased although LLRs produced by C-ion beam have not determined yet. In this study, Syrian golden hamster embryonic (SHE) cells were irradiated with C-ion beam at HIMAC as LET: 13.3 keV/μm, 1 kGy, 0.477 kGy/h. Other SHE cells were also γ-irradiated with same dose and dose rate, then the ESR spectra on C-ion and γ-ray radiation were compared. Not only SLF radicals but also thyl (TYL: R-S&bull) radicals were produced by either C-ion and γ-ray radiation. Addition of RibCys to both irradiated cells scavenge SLF radicals, but TYL radicals were scarcely decreased. It indicates that SLF radicals could be mutagenic. A SLF radical is produced by the reaction between a cysteine and a O₂— molecule during radiation and is scavenged by Vit. C, RibCys, epigallocatechin-3-O-gallate, N-acetylcysteine, GSH, and so, those chemicals commonly have hydrophilic and hydrogen reducing characters. These results indicate that mutagenic SLF radicals may be generated on the surface of some proteins but TYL radicals may exist in interior of the proteins where is hydrophobic.

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We have previously found that in A-survivors who were exposed in utero, only a small increase in the translocation frequency was observed in T lymphocytes (examined at age 40). To clarify the underlying mechanisms, we conducted studies in mice. Pregnant mice (15.5th day p.c.) or mice of various ages were irradiated with 2 Gy of X-rays. When the mice reached the age of 20 weeks, translocation frequencies (FG) were measured with FISH in blood T cells, spleen T cells, and bone marrow (BM) cells. To test the possible involvement of apoptotic elimination of chromosomally damaged cells, fetal mice of p53-/- or p53+/- were also irradiated and examined. FG at 20 weeks of age was very low if mice were irradiated in utero or soon after birth as we have seen in A-survivors, but it gradually increased with the increase of age at the time of exposure, and finally reached the same level of adults after 6 weeks of the age. p53 gene status did not affect the low FG in irradiated fetuses. We also examined acute radiation effect 4 to 48 hrs after the exposure in spleen cells from 4-day old mice, and found many chromatid-type aberrations as we usually find in BM cells from adults. We interpreted the results as indicating that progeny cells from survived stem cells after irradiation, which are aberration free by some reasons in fetuses or neonates, washed out the aberration-bearing lymphoid cells during the growth period. The apparent radioresistant nature of the stem cells is not related to p53 gene status but the underlying mechanisms are not clear as yet.

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We examined the DNA double strand break (DSB)/chromosome repair in human cells irradiated with high LET radiation. Normal human fibroblasts (HFLIII) were irradiated with carbon ion, neon ion, and iron ion radiation. After 0, 0.5, 1, 2, 6, 24 hours, the DSB repair process was monitored with the G1-type premature chromosome condensation (PCC) technique. Fluorescence in situ hybridization (FISH) was also performed with the PCC samples (24hours after irradiation). Although cells were exposed to identical LET (70keV/μm) as carbon ions, the repair process of cells irradiated with neon ions was slower than with carbon ions. The remaining number of fragments for neon irradiation was much lower than with iron ions (LET: 200keV/μm). After 24 hours from irradiation with neon ions, cells had almost repaired similar to cells irradiated with X-ray and carbon ions but cells irradiated with iron ions had unrepairable or difficult DSB. FISH assays are ongoing. Our results with G1 PCC indicate that different ions that have equal LET values can imply different repair process.

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It is becoming clear that ionizing radiation causes cell death and chromosome aberration not only in directly irradiated cells but in non-irradiated cells surrounding the exposed cells, whose phenomena are called bystander effects. Some factors secreted into the culture medium have been thought to be involved in the induction of bystander effects, but the mechanism is not clear. We aimed to identify bystander factors in normal human diploid cells exposed to X-rays. The experiments are performed with preparing two 60X24 mm cover slips, on which 2X10⁴ cells are attached. The cells on one cover slip are irradiated with X-rays as donor cells, while the other cells are co-cultured with donor cells as recipient cells. We analyzed cell growth and micronuclei induction in the recipient cells. Twenty-four hours after irradiation, the number of recipient cells was similar to that of the control ((1.17±0.11)X10⁵). In contrast, the frequency of micronuclei observed in recipient cells were (7.0±1.5)X10^-3, and control were (3.2±0.3)X10^-3. It was two-fold higher than that of the control. Furthermore, we found the frequency of micronuclei was independent of the donor cell's number, or doses. Because ROS and NOS are suggested to be involved in the induction of bystander effects, we examined the effect of DMSO on the induction of micronuclei in the recipient cells. However, there was no suppression, indicating that not short-lived ROS, but long-lived ROS or NOS may be involved in bystander effects.

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Clonal chromosome aberrations were detected by scoring 5,000 blood lymphocytes from each of five atomic bomb survivors whose observed translocation frequencies were 6% to 12% with FISH using probes for whole chromosomes 1, 2, 4. After FISH screening of translocations involving the painted chromosomes, all the slides were re-stained for Q-banding to determine the unpainted, counterpart chromosomes and the breakpoints involved in the translocations to identify clonal aberrations (i.e., identical aberrations carried by at least three cells). The minimum clone size detected was 3/5,000 or 0.06%. Among 2,444 aberrant cells observed, 44 different clones were detected. Specifically, we found 31 clones (clone size; 3-5 cells/5,000 or 0.06-0.11%), 5 clones (0.12-0.23%), 2 clones (0.24-0.47%), 2 clones (0.48-0.95%), one clone (0.96-1.91%), 2 clones (1.92-3.83%), and one clone (3.84-7.58%). The number of clones was inversely related to the clone size as we found previously by scoring 500 cells from about 500 survivors (detectable clone size ≥0.6%). The results indicate that the clonally derived lymphocytes, whether chromosomally marked or not, are ubiquitous. In other words, a fraction of normal cells are also clonally derived, and they lessen the frequency of non-clonal translocations (bio-indicator of radiation dose) by 5% to 10% collectively and further down by as much as 50% in some individuals.

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It is difficult to determine whether SCID mice are susceptible to radiation-induced tumors other than thymic lymphomas (TL) , because SCID mice are extremely sensitive to TL induction by irradiation with a short latency period. SCID mice having a mutation on DNA‐PKcs gene exhibit the limited activity on repair of DNA double strand breaks, and not only blood cells but also somatic cells are sensitive to the lethal effect of radiation. To study the relationship between scidmutation and tumorigenesis, thymic lymphomagenesis in female SCID mice was suppressed by iv injection with bone marrow cells of wild-type male mice following whole body irradiation of 0 to 3Gy γrays. Fifty to100 mice per group were used in each group and resultant tumors were analyzed. The replacement rate of bone marrow cells and thymocytes were determined by FISH method with Y chromosome-specific probe. When thymic lymphomagenesis was suppressed by transplantation with wild-type bone marrow cells, the tumor incidences in SCID mice were the same or lower than those of wild‐type mice with the same treatments. Thus there was no induction of solid tumors by radiation in SCID mice. This suggests that a defect in nonhomologous endjoining repair does not contribute to the development of solid tumors.

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ICRP has stated that any life-shortening found in exposed human populations and in experimental animals after low doses has been shown to be due to excess radiation-induced cancer mortality (Publication No.60, 1990). The tissue weighting factors to define the quantity effective dose are based on the detriment which includes the probability of attributable fatal cancer, the weighted probability of attributable non-fatal cancer, the weighted probability of severe hereditary effects and the relative length of life lost. One of the authors has proposed a formula to estimate a contribution of cause of death to life shortening (J.Radiat.Res.20, 284-290,1979). A total of 4000 B6C3F1 SPF mice were used for study on late effects of chronic exposure with gamma-rays at low dose rates. The dose rates were 0.05, 1.1 or 21 mGy/day and the accumulated doses were 0.02, 0.4 or 8 Gy respectively (irradiation period of approximately 400 days). Statistically significant life shortening was observed in 0.4 Gy group of female mice and in 8 Gy groups of both sexes The contributions of causes of death were calculated with the method quoted above. Large 3 contributions were made from malignant lymphomas, non-tumor diseases and soft tissue tumors in 0.4 Gy and 8 Gy groups of female mice. In 8 Gy group of male mice, large 3 contributions were made from malignant lymphomas, non-tumor diseases and hemangiosarcomas. These contributions were mainly due to earlier appearance of the causes of death instead of increase in mortality. The work was supported by grants from Aomori prefecture.

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To prove that mutation in the p53 gene is the primary cause of cancer, tumor induction was tested with transgenic mice bearing TP53 mutation. The cDNA of p53 with 9 bp deletion at exon 6 was obtained from radiation-induced mouse tumors, and cloned into the expression vector pCXN2 (named pTE50). pTE50 was introduced into embryos of C57BL/6 mice and one male transgenic mouse with the p53 mutation was obtained, as confirmed by PCR and Southern blot analysis for the existence of the mutation in mouse genome and by mRNA selective PCR for its expression. Mice were injected with 0.1 ml of 20-methylcholanthrene (0.2 mg/ml dissolved in olive oil) at the right groin, and were followed for tumor induction for one year. Male mice with the mutant p53 (+/-) showed a high tumor induction rate (fibrosarcomas, 90% at present). Female mice showed a lower tumor induction rate (under examination). Control mice, males and females, showed a tumor induction rate lower then 30%. These results provide a direct evidence that mutation in the p53 gene is an initial trigger of tumorigenesis.

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We have reported that the 5'-deletions of Notch1 gene are formed by the illegitimate V(D)J recombination or the microhomology-mediated nonhomologous end-joining (MNHEJ) in radiation-induced mouse thymic lymphomas. Our previous study showed that the illegitimate V(D)J recombination occurs spontaneously in mouse thymocytes and radiation does not induce the illegitimate V(D)J recombination. To determine the spontaneity and the induction of the MNHEJ by radiation in mouse thymocytes, thymus DNA was isolated from unirradiated or irradiated wild-type mice and the abnormalities of Notch1 gene from position 4,758 to position 16,800 were examined by nested PCR. In unirradiated mice, 28 Notch1 deletions were detected in 8 x 10⁶ thymocytes of four mice, of which 11 were formed by the illegitimated V(D)J recombination, three were by the MNHEJ, and 10 were by unknown pathway. Thus both the illegitimate V(D)J recombination and the MNHEJ occur spontaneously in thymocytes of wild-type mice. In mice irradiated at1.6 Gy γ-rays four times at one-week intervals, the frequency of deletions formed by the MNHEJ did not increase, suggesting that the MNHEJ pathway is not induced by radiation in thymuses of wild-type mice.

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Aberrant methylation of CpG islands, which are CpG dinucleotide rich areas located in the promoter region of many genes, serves as a mechanism for inactivation of tumor suppressor genes in many cancers. Recently, it is reported that lung tumors induced by plutonium and, to a lesser extent, X-rays showed higher frequency of estrogen receptor (ER) promoter than those induced by NNK, suggesting a close link between radiation exposure and promoter methylation. In the present study, we examined the status of promoter methylation of tumor-related genes in radiation-induced rat mammary tumors, comparing with spontaneous and MNU-induced ones.DNA from spontaneous, MNU (40mg/kg)-induced and gamma-ray(2Gy)-induced mammary tumors in SD female rats were modified with bisulfite. DNA sequences in promoter region of E-cadherin (Cdh1), Rassf1, p16 (Cdkn2a), Apc, ER (Esr1) were amplified by sets of primers, which do not contain CpG dinucleotide. PCR products were then subjected to TA cloning and DNA sequencing. Promoter of Cdh1 was slightly methylated in gamma-ray-induced tumors, but rarely methylated in spontaneous and MNU-induced ones. Methylated CpG scattered over the CpG island in the p16 promoter regardless of the carcinogens, but densely methylated sequence was rarely obserevd. Contrary to the evidences of heavily methylated promoters of CDH1 and P16 in human breast cancers, our preliminary results suggest that DNA methylation does not play a critical role in the inactivation of tumor suppressor genes in rat mammary tumorigenesis.

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Irradiation protocol for thymic lymphoma development includes 2.5Gy γ-ray exposure four times at a weekly interval, and this leads to the persistent atrophy of thymus, some developmenting lymphoma. Microenvironment in this atrophic thymus is thought to be important for lymphomagenesis. It was reported that "prelymphoma cells " already exist in the atrophic thymus as early as one month after irradiation. We produced atrophic thymuses by applying this irradiation protocol to Rit1/Bcl11b wild-type and heterozygous mice. Rit1 is a novel tumor suppressor gene on mouse chromosome 12. At first, clonality of thymocytes was analyzed by determining DNA rearrangements in D-J recombination sites within the TCRßgene. The thymuses of both genotypes comprised less than one tenth of cells and showed only a few fragments, indicating thymocytes of mono- or oligo-clonal origin. LOH at the Rit1 and Ikaros loci was then examined. LOH was found at the Rit1 locus at a high frequently but very low at Ikaros, irrespective of the genotype. The results strongly suggest that atrophic thymuses contained pre-malignant cells of clonal growth that underwent genetic alteration of Rit1 but not yet of Ikaros. Other loci including p53, Myc, and Notch1 were also examined. We will show our findings, which indicate genetic changes in this order: unknown gene X, Rit1, (p53, Myc and Notch1), and Ikaros.

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High-dose rate radiation induces acute myeloid leukemia (AML) in C3H mice. The proliferated AML cells in spleen have deletion of chromosome 2 in 92.4%, where associates 50-100 Mb hemizygous deletion of particular region nearby D2Mit15 marker, involving PU.1 gene. PU.1 is a transcriptional factor for hematopoietic differentiation and is considered to be related with development of AML. PU.1 protein promotes the expression of receptors G-CSF, GM-CSF and M-CSF. It is speculated that chromosome 2 deletion containing PU.1 gene occurred by radiation decreases PU.1 expression, and it induces inhibition of hematopoietic differentiation, finally it resulted in AML development. In present study, we analyzed whole genome mutations by using array CGH. in AML cells induced by high dose rate gamma rays (0.5Gy/min, 3Gy). 695 BAC clones mostly had been nearby oncogenes for analysis. All of the AML samples identified deletion of chromosome 2 by FISH, using a BAC probe containing D2Mit15. deletion on chr.2 positive-AML had additional aberrations mostly on chromosome6 and X, on the while deletion chr.2 negative AML had aberrations on only chromosome X, but 6 was not. The different genome patterns in both AMLs indicate that two different pathways in hematopoietic differentiation associate with development of these AMLs induced by high-dose irradiation. The information will be important for comparison of genome alterations with AML cells induced by low dose rate radiation. This study was supported by a grant from Aomori Prefecture.

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Chromosome analyses were performed on mouse thymic lymphomas (TL)induced by carbon beam. A total of 12 specimens (B6: 5 specimens, C3H: 6 specimens and F1 : 1 specimen) were analyzed by Quinacrine-Hoechst banding technique and chromosome painting method. Trisomy of chromosome 15 was observed in all specimens from C3H mice and F1 mouse, but not identified in TL from B6 mice. On the other hand, the abnormality derived from chromosome 11 was identified in four specimens from B6 mice. Furthermore, the abnormality of chromosome 7 was also detected in three specimens from B6 mice. Thus, the chromosome abnormality in mouse thymic lymphoma induced by carbon beam was not identical between these mouse strain. These results in the present study indicate that the genetic background of the host animals may have a profound influence upon the genetic event in the development of thymic lymphoma by carbon beam.

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X-ray induction of tumors was examined in five-week-old male OLETE rats, treated with two 10Gy doses to the gastric region with a 3-day interval (total 20Gy). After irradiation, rats were received in commercial diet MF and tap water and kept 564 days after the first X-irradiation. The mean serum glucose level of the X-irradiated group was significantly lower than that of non-irradiated group 18 month after first X-irradiation. Total tumor incidence was 27/30 (87%), the first animal demonstrating a gastric and pancreatic islet tumors in X-irradiated group and 6/19 (32%) in non-irradiated group. The islet tumors developed in 18 rats (60%). Immunohistochemistry revealed in the islet tumor cells to be generally positive for insulin. Furthermore, rats with pancreatic tumors had lower serum glucose. The present results thus showed that the insulinoma are induced by X-irradiation in OLETE rats.

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[Objective] Epidemiology on A-bomb survivors has clarified that childhood exposure confers a high breast cancer risk. But the underlying mechanism is unclear. Using Sprague-Dawley rats, we attempted to verify if childhood exposure (1) results in increased development of mammary cancer and (2) affects susceptibility to a subsequent carcinogen.
[Methods] (1) Rats were γ-irradiated (2 Gy) at 3 or 7 weeks (w) of age. (2) Non-irradiated rats and rats irradiated at 3 w were treated with 1-methyl-1-nitrosourea (MNU; 20 mg/kg, i.p.) at either 3 or 7 w. They were observed until 50 w.
[Results] (1) Spontaneous palpable mammary cancer multiplicity was 0.07 ± 0.04 (tumors/rat, mean ± SEM). Rats irradiated at 3 w developed significantly less tumors than those irradiated at 7 w (0.28 ± 0.11 vs. 0.90 ± 0.24, respectively). (2) Contrarily, rats treated with MNU at 3 w developed significantly more tumors than those treated at 7 w (0.56 ± 0.20 vs. 0.11 ± 0.08, respectively). After irradiation at 3 w, MNU treatment at 3 w resulted in an additive effect (0.76 ± 0.20), while treatment at 7 w elicited a synergistic effect (0.71 ± 0.18).
[Discussion] Relatively low susceptibility to mammary cancers after prepubertal radiation exposure in rats contradicts with epidemiologic data on A-bomb survivors. However, our data suggest that childhood exposure deposits a long-term effect that becomes apparent in combination with subsequent carcinogen exposure. The nature of this long-term effect remains to be elucidated.

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Dysregulation of cytokine receptor expression and downstream signal transduction cascade play important roles in lymphomagenesis. In this study, we examined expression of cytokine receptors (IL-2R, 4R, 7R, 9R and 15R) and the activation status of downstream STAT pathways in mouse radiation-induced thymic lymphoma (TL) cells. TL was induced by exposure of TL-susceptible C57BL/6N(B6) mice and TL-resistant C3H/HeN(C3H) mice to split-dose X-irradiation (1.6Gy x 4 times). Expression of cytokine receptors was examined by semi-quantitative RT-PCR and western blotting. The results obtained here were as follows: (1) TL from B6 mice showed dramatically high expression of IL-9Ralpha compared to normal thymocytes by 60 folds, (2) IL-9Ralpha protein was also abundantly expressed and (3) high activation of STAT3 and STAT5, but not STAT1, were manifested in TL expressing high amount of IL-9Ralpha. (4) In contrast to TL from B6, TL from C3H showed just 3-fold expression of IL-9Ralpha compared to normal thymocytes. Activation of STATs was also much less compared to B6 TL. These findings suggested a correlation between the constitutive activation of STATs possibly resulting from IL-9Ralpha over-expression and the TL-susceptibility in mice. Investigations of phosphorylation of IL-9Ralpha, and activation of JAK1, which is associated with IL-9alpha, are in progress.

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BALB/c (C) mice are susceptible to radiation-induced lymphomagenesis, while STS (S) mice are resistant. Previously, by analyzing (CxS)F₁ x C mice we mapped two loci responsible for resistance to radiation-induced lymphomagenesis in the vicinity of D4Mit17 and in the D4Mit302-D4Mit255 region on STS-derived chromosome 4. In the present study, a series of C.S congenic lines carrying the STS allele at either of the two loci or in the regions neighboring them on chromosome 4 in the BALB/c background was selected and examined. Animals were exposed to 4 x 1.7 Gy of X rays and development of lymphomas was observed during 300 days after irradiation. Genotyping of the C.S lines was carried out with PCR and PAGE. Survivals of the C.S. lines were compared with that of BALB/c using the Kaplan-Meier method. The STS allele in both of the D4Mit322-D4Mit86 and D4Mit302-Cdkn2a segments conferred resistance to lymphomagenesis, while the STS allele in the segments D4Mit39-D4Mit163, D4Mit86-D4Mit302 and D4Mit255-D4Mit31 did not. The results indicate the presence of a novel resistant gene for lymphomagenesis in a 10 Mb D4Mit7-D4Mit86 region with syntenic homology to human 9q32-33 containing a candidate tumor suppressor gene DBCCR1.

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Radiation carcinogenesis in human is considered as a result of the combined effect of radiation and environment factors. We previously showed that smaller doses of X-rays reduced the incidence of TL induced by N-ethyl-N-nitrosourea (ENU), while large doses of X-rays increased and accelerated the TL development in a synergistic manner. This indicates the mode of combined effect is dependent upon the dose of X-rays. In this study, we aimed to determine the mode of combined effect on tumors other than TL and life-span (LS) shortening.
B6C3F1 mice (4-week-old female) were irradiated with X-rays (0.2 - 2.0 Gy a week x 4 times) or treated with ENU for 4weeks (50 - 400 ppm in drinking water) from 8-week-old. For combined treatment, the mice were irradiated with X-rays (0.2 - 1.0 Gy a week x 4 times) and then treated with ENU (50 - 200 ppm).
The LS after combined exposure (0.2/0.4 Gy X-rays with 200 ppm ENU) was much longer than that of ENU treatment alone, suggesting small dose of X-rays has antagonistic function. Contrary to the results of TL, incidence of tumors of lung, liver and ovaries increased more than additively after the combination of ENU with X-rays even at small doses. Thus, the role of X-rays in combined exposure was dependent not only the dose used but also the tissues examined.

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Ikaros is a potent tumor suppressor gene for both human and murine leukemogenesis. Ikaros alteration in X-ray-induced thymic lymphoma (TL) was characterized by aberrant splicing, null expression and point mutation, which were accompanied by loss of heterozygosity (LOH) at Ikaros loci. N-ethyl-N-nitrosourea (ENU) -induced TL harbored only point mutation without LOH. We showed that combined treatment of X-rays (0.8-1.0 Gy x 4 times) and ENU (100, 200ppm) synergistically increased TL incidence. The aim of this study was to elucidate the underlying mechanism of the lymphomagenesis induced by combined treatment.
B6C3F1 mice (4-week-old female) were irradiated with X-rays (0.2 - 1.0 Gy a week x 4 times) and then treated with ENU (50 - 200 ppm in drinking water) from 8-week-old. The analyses of LOH, expression and mutation of Ikaros were performed.
The frequency of aberrant splicing, null expression and point mutation of Ikaros in X-ray-induced TL was 10, 12 and 26%, respectively. ENU-induced TL showed only point mutation in 26% of TL. LOH frequencies at Ikaros loci were 73% and 0% for X-ray and ENU-induced TL, respectively.TL after combined treatment rarely exhibited aberrant splicing and null expression. Surprisingly, point mutation was increased up to 40%, most of which did not accompany LOH, indicating similarity to those in ENU-induced TL.
We conclude that X-rays play a role as a promoter for ENU-induced lymphomagenesis in combined exposure.

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In evaluating cancer risk of neutrons, experimental data on energy dependency of RBE are needed. We started a series of experiments on murine carcinogenicity, especially focusing on leukaemogenesis, of low energy range (0.5MeV- 10MeV) of neutrons. In the first experiment, to evaluate the carcinogenicity of 10MeV neutron, male C3H/nrs mice, susceptible strain to radiation-induced leukemia, were exposed to neutrons generated from cyclotron, using d (25 MeV)-Be reaction at the peak energy of 10 MeV (dose rage: 0.05 - 2Gy ) or to Cs-137 gamma-rays (dose range: 0.2 - 4Gy). The animals were under observation for their life span. Life-shortening effect was significant at more than 1Gy of gamma-rays, and at more than 0.2Gy of neutrons. RBE was calculated to be about 4. The incidence of myeloid leukemia after exposure to neutron- and gamma-rays mice increased linearly in dose dependent manner. RBE was calculated to be about 1.5. RBE of Harderian gland tumor was calculated to be about 8.

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To clarify the potential role of high LET radiation-induced carcinogenesis in respiratory tract, the cytotoxicity and transforming ability of gamma-rays and alpha-particles were analyzed utilizing primary cultures of rat lung (RLE) and tracheal epithelial (RTE) cells. The radiation sensitivity and relative biological effectiveness (RBE) for alpha-particles in RLE and RTE cells were compared. The irradiation caused similarly an exponential decrease in survival in RLE and RTE cells, and the D37 of alpha-particles and gamma-rays were 0.65 Gy and 3.6 Gy, respectively. The RBE for cell killing was 5.5 in the both types of cell. The transformation frequencies (TF) for RLE and RTE were 3.7x10^-3 and 2.4x10^-3 at 2 Gy of alpha-particles, respectively. At 7.5 Gy of gamma-rays, the TF for RLE and RTE increased to 8.0x10^-3 and 7.1x10^-3, respectively. The RBE for transformation of RLE was 1.7, and that of RTE was 1.3. This primary epithelial cell culture system will be useful for analysis of radiation sensitivity among the different target cells and mechanistic studies of early changes in the rat lung carcinogenesis induced by alpha-particles.

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Ionizing radiation induces DNA double-strand breaks, which result in cell death, if they are not repaired. Surviving cells repaired DNA damage, and they are expected to grow normaly, however, it is becoming clear that delayed reproductive death, delayed chromosomal aberrations, and delayed mutations are frequently observed in the progeny of surviving cells. These phenomenon called radiation-induced genomic instability in the progeny of surviving cells are studied extensively, but the mechanism underlying is not clear.We have hypothesized that DNA double-strand breaks may create potentially unstable chromosome regions (PUCR) at the broken sites through DNA double-strand break repair, and that PUCRs could be reactivated when chromatin structure is changed during DNA replication, or through gene expression. To prove the hypothesis, GM 638 cells (SV40-immortalized cells) are irradiated with 3 Gy of X rays, and the primary colonies having large deletions at the HPRT locus on X chromosome are isolated. The primary colonies are seeded again, and the frequency of colonies with giant cells were examined. We found that colonies with significant numbers of giant cells were detected about 7% among the secondary colonies. It is suggested that the secondary change may arise at PUCRs, and reactivated PUCRs could cause giant cells within the secondary colonies.

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Lethal Sectoring, Genomic Instability, and Delayed Division Delay in HeLa S3 Cells Surviving Alpha- or X-irradiation*Hiroshi SASAKI Dept. Exp. Radiol., Kyushu Univ. Lethal sectoring is the process for survival in which lethal damage remaining in irradiated cells is eliminated as offspring without reproductive integrity. This process occurs through the postirradiation 1st to 4th divisions with the accompanying appearance of a clonogenic progenitor. The features of lethal sectoring and delayed cell death were explored by analyzing the pedigrees of HeLa cells surviving alpha- (0.45 Gy) or X-irradiation (3 Gy) (20% survival dose). Most (ca.70%) of the lethal damage was eliminated from alpha-particle survivors through the 1st to 2nd divisions, but it persisted in X-ray survivors until the 2nd generation. Nonlethal damage remaining in the clonogenic progenitors led to an elevated incidence of delayed cell death in their progeny. The mean incidence was higher for alpha-particle (16.3%) than X-ray survivors (8.3%), indicating the greater potentiality for genomic instability (GI) by alpha-particles. Misrepaired clustered DNA damage by alpha-particles and unrepaired PLD by X-rays seemed to be involved in the induction of GI. Delayed division delay (DDD) was noticed, though occasionally (ca.10% per cell), with the progeny during the postirradiation 1st-3rd generations. Mean DDD was much longer in alpha- (ca.11 h) than X-irradiated cells (ca.4 h). Supposedly DDD was triggered by delayed chromosome breakage (published in JRR 45: 497-508 [2004]).

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When we think about genetic influence of radiation, non-targeted effects of radiation is an important object. And we have to leave a conventional paradigm that a target of genetic effect of radiation is DNA. Influence of radiation does not appear as direct influence of radiation. Influence of radiation appears away from initial hit both in terms of time and space. This phenomenon is called genetic instability. However, induction mechanism is not made clear. This study planned to clarify how a p53 gene function contributed to genetic instability derivation. In this study, we used a primary culture cells derived from p53 gene normal (+/+) and knockout (-/-) C57B mouse. 10⁶ cells were inoculated into T75 flask and each culture was subcultured at every 5day. As a result, the p53 normal cells grew with a growth rate 1 until passage 6 or 7 generations. However, the cells restored proliferation potency again when passage reached at 10. On the other hand, p53 knockout cells continued multiplying at growth rate 3.1-.53 lively without passing through temporary breeding degradation. Furthermore, we analyzed a change of chromosomal karyotype with increase of passage. The structural aberration was not seen in normal and knockout cells. However, we found that 35-40% of p53 knockout cells have 88 chromosomes. This means that p53 knockout cell is defective in chromosomal homeostasis maintenance mechanim. This shows that p53 plays an important role in chromosomal homeostasis maintenance.

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Mouse genome possesses 2000 copies of intracisternal A-particle (IAP) DNA element. From the element, shorter mRNA is transcribed and the complete cDNA is synthesized by an unique mechanism closely related to retrovirus life-cycle. By an integration of the cDNA into genomic DNA, adjacent gene function is influenced. We have previously found that genomic rearrangement by characteristic insertions of new copies of the IAP cDNA frequently occurs in acute myeloid leukemia cells of C3H/He mouse. Since there are large amounts of IAP-related DNA/RNA molecules in any of mouse cells, it is difficult to analyze the complete IAP cDNA that has the potential to convert genomic information.
To reveal the unique reverse-transcription, we designed a series of IAP genomes for quantification of complete cDNA derived from the RNA molecules. The synthesized IAP genomes were stably integrated to mouse macrophage cell line, RAW264.7. A faint amount of the cDNA from the transgene RNA was successfully measured by real-time PCR in the transformants. Surprisingly, the level of the cDNA was increased in the cells following to exposure to ionizing radiation. This suggests that the radiation not only damages genomic DNA but also stimulates chronic endogenous mutation via IAP cDNA.

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The C. elegans develops through four larval to reach adult.However, under adverse environmental conditions, worms will enter the dauer.If the environmental condition improves during the dauer, the worms will recover to adult.Interestingly, the duration of dauer has no effect on the postdauer life span. Showing strong resistancy to stress such as oxygen, UV and high temperature, it is thought that dauer larvae have some efficient repair and riddance mechanisms against the damages at the dauer. As the result, it seems to have no effect on postdauer life span. However, the studies related to the stress response and aging in dauer larva have not been done yet. Then, aim of this study is to elucidate the repair and riddance mechanisms of dauer larva against the damages. To analyze the behavior of damage during dauer, Dauer larvae were exposed to X rays of 100,300 and 500 Gy at different times of dauer, and we measured the recovery rate and postdauer life span after recovering. The result showed that the recovery rate decreased with increase of X ray dose. We observed that dauer irradiated 100 Gy extended the postdauer life span compared to control worms. This is thought to be due to the hormesis effect of dauer larvae. Moreover, X ray damage at the early stage of dauer did not influence the postdauer life span. On the other hand, the effect on the postdauer life span of the worms which were recovered just after irradiation were shorten independent on duration in dauer. It is the first report that abilities of repair and riddance also impaired with aging during the dauer.

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Pregnant females of C57BL/10J mice at 9 days of gestation were whole-body irradiated with a single acute dose of silicon-ion-radiations. The effect was studied by scoring changes in the prenatal and postnatal development of mice as well as in the pigmentation in prenatal hair follicles and cutaneous coats 22 days after birth. The frequency of abnormalities in the fore and hind legs, tails and eyes as well as of hemorrhage was increased as dose increased and the number of embryos as well as the body weight of the 18-day-old embryos was decreased. The percentage of birth, the survival to day 22 and the body weight at day 22 were also reduced. By comparing the survival to day 22 for silicon-ion-radiations with that of gamma-rays, silicon-ion-radiations were more than twice as effective as gamma-rays. In 18-day-old embryos, the development of hair follicles was delayed as dose increased. The frequency and the size of the white spots in the mid-ventrum were increased in the irradiated mice. Silicon-ion-radiations were more effective than gamma-rays. These results suggest that silicon-ion-radiations have a greater effect on prenatal and postnatal development of mice as well as on the melanocyte development than gamma-rays.

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Epigallocatechin gallate (EGCg) has been known to be a powerful anti-oxidant and a radical scavenger. However, its role of proliferation and radioprotection has remained to be unclear. Therefore, we investigated that the effects of EGCg on megakaryocyte progenitor cells which carried out radiation exposure using CD34⁺ cells prepared from the human placental and umbilical cord blood.
The colony forming unit-megakaryocyte (CFU-Meg) were assayed with thrombopoietin (TPO) alone or together with EGCg by plasma clot technique using platelet poor human plasma. EGCg showed the dose-dependent efficacy of increasing the CFU-Meg number up to 100 nM. EGCg stimulated a 1.5-fold increase in the total CFU-Meg number compared with the control. In the culture of 2 Gy-irradiated CD34⁺ cells, the similar increasing effect was observed by the addition of EGCg. Especially, its radioprotective effect is shown at a relative low dose (0.5 - 2 Gy) from the radiation survival curve.
Non-irradiated and X-irradiated (2 Gy) CD34⁺ cells were cultured in liquid culture medium contained with TPO alone or TPO plus EGCg for 14 days. The combination of TPO plus EGCg resulted in increase in the CFU-Meg number in the both of 0 Gy and 2 Gy.
These results suggest that EGCg shows the radioprotective effect on the growth of CFU-Meg at low dose of X-irradiation, and it is possible to use as one of radioprotective agents.

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α-amylase (EC 3.2.1.1) is a digestive enzyme, and is mainly a constituent of pancreatic juice and saliva. Irradiation is known to increase the activities of α-amylase in serum. However, the mechanism is not fully understood. Irradiation increased the α-amylase activity in plasma with the concomitant decreased activity in saliva of mice in a dose-dependent manner. Irradiation did not affect the levels of α-amylase transcripts in the human salivary gland cell line HSY. Therefore, we prepared acinar cells from the parotid glands of mice and cultured them in vitro. Irradiation decreased the α-amylase activity in these primarily cultured cells and increased the activity in the supernatants. Confocal fluorescence images of cells stained with anti-amylase antibody showed that irradiation decreased the fluorescence intensities. Furthermore, horseradish peroxidase (HRP) was administered retrogradely via the main excretory duct of parotid gland in a rat as a tracer. The tracer was present in cells from an irradiated rat but not in control. When HRP was injected intravenously, the tight junction prevented the passage of tracer from acinar lumina and intracellular canaliculi into the intercellular space in control. However, the tracer was detected at these areas in an irradiated rat. Our results suggest that irradiation may cause increased levels of α-amylase in blood by not altering the production of amylase but affecting barrier function; amylase is released into blood vessels through distracted barrier by irradiation.

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The developing brain is known to be one of the fetal structures most susceptible to radiation. The neurons of the cerebral cortex are distributed in an inside-out manner, with early generated cells forming deeper layers and subsequently born neurons occupying successively more superficial positions. To study the effect of low-dose prenatal X-irradiation on the cortical development, we developed a birthdate-related fluorescent labeling technique and a live-imaging technique of brain slices. In this study, we subjected 0.5-2.0 Gy of X-irradiation on mouse embryos. And then, we visualized and distinguished the cohort of birthdate-related cortical neurons from other neurons with different birthdates in the embryonic brain slices on a fluorescence microscope. In the control brain slices, neurobrast in ventricular zone produced the multipolar cells in intermediate zone (IZ). Those cells turned to bipolar cells and migrated toward the marginal zone (MZ), and anchored the leading process. Then, the leading processes were changed to apical dendrite, and cell bodies moved downwards and lined up. Whereas, in the X-irradiated brain slices, the migrating cells from IZ toward the MZ had some branched processes, and sometimes stopped during the migration. Some of the other cells seemed to have abnormal apical dendrite and not to line up. These observations suggest that the low-dose X-irradiation affects several aspects of neuronal migration in mouse cortical development.

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Since the classic discovery of the mutagenic effects of X-ray in Drosophila melanogaster by Muller (1927), ionizing radiation, including &gamma-ray, has been a major tool for geneticists. The irradiation effects on mutation in Drosophila have been shown to be dependent on total dose and independent of dose rate. Russell et al. first observed that the effects of radiation depended not only dose but dose rate in the spermatogonial stem cell mutation frequency. We observed dose-rate effect on micronucleus formation and proliferation inhibition of human cells, and established a novel dose-rate effect model, MOE model. Here we study the dose rate effect on lethality of Drosophila, which has provided basic concepts of radiation biology. Third-inster larvae were irradiated with &gamma-ray at various dose-rates for constant irradiation time up to three days in an irradiation room bearing 50,000 Ci ⁶⁰Co. The LD₅₀ for irradiation time less than one hour was constantly about 46 Gy, while those for irradiation time longer than one hour gradually increased. The LD₅₀ for 70 hour irradiation was more than triple of that for short irradiation times. They are linearly correlated to logarithm of irradiation time. We also found that observed data followed MOE model, when logarithm the LD₅₀ was plotted against logarithm dose-rate. These results suggest that lethal effect of ionizing radiation on Drosophila has dose-rate effect at low dose-rate, and that this dose-rate effect is well described with MOE model.

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Life span study was performed to examine the life expectancy, incidence of tumors, and bone fragility after heavy ion particle (HIP) exposure to the whole body of mature rats, as models of adults of low radiation sensitivity compared to young. Female Wistar (WM strain ) rats, 12 months old, normal and with no mammary tumors, were irradiated with the doses of 0.5, 0.75, and 1.0 Gy by HIP radiation (Carbon 290 MeV) and 0.5, 1.0 Gy by X-ray (n= 30~50 rats). The rats were kept until they died. The anatomical and histological findings were obtained. The average life expectancy in all radiation groups was significantly shorter than that of the control. The incidences of mammary tumors in the HIP groups increased (20.0~37.5%) in a dose-dependent manner to the doses, and were higher than those of X ray groups (13.3~20%), although the control group was 12%. Also, the incidences of ovary and lung tumors in the HIP groups were higher than that of control group. Pituitary damages increased at 0.5 Gy of HIP group, but decreased at 0.75 and 1.0Gy, than those of X-ray and control groups. There were no significant differences in the bone mineral density of tibia between the radiation groups and control group. The results indicted that whole body exposure of HIP radiation might induce shortening of life expectancy, increase the incidence of mammary and ovary tumors in rats, even if exposed at the mature age.

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The regional irradiation with carbon ions induced bone responses that were qualitatively and quantitatively different from those induced by gamma irradiation at an equivalent dose. Irradiation with carbon ions resulted in a dose-dependent increase in bone volume, while gamma irradiation induced a loss of bone volume. We examined how carbon-ion irradiation of the osteoblastic cell line MC3T3-E1 influences proliferation and differentiation. The mRNA expression levels of the osteoblast proliferation and differentiation factors cbfa1 and MMP13 after carbon-ion and gamma irradiation were also compared. Gamma-irradiated cells had increased MMP13 levels. The mRNA expression levels of cbfa1 in carbon ion group had not changed significantly, but decreased cbfa1 expression in gamma ray group. Those finding suggest the possibility of reflecting the carbon ion resulted increase in bone volume.

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The objective of the present study is to examine possible protective or treatment methods for brain tissue injury induced by charged ion particles. Male Sprague-Dawley rats aged 10-14 weeks were used and their left cerebral hemispheres were irradiated at a dose of 100Gy with charged carbon particles (290 MeV/nucleon, 5mm spread-out Bragg peak). In order to observe the protective effects of a free radical scavenger on brain tissue injury, some rats were injected intraperitoneally with 400 mg/kg radcut just before irradiation. The gross anatomy of the brain was examined at different times after irradiation using a clinical magnetic resonance imaging system. Histological and behavioral changes over time were observed and compared with magnetic resonance histological changes like necrosis, vascular dilatation and tissue swelling at the irradiated region were induced at 8 week after irradiation, and such changes could be identified with magnetic resonance imaging. Behavioral changes in walking patterns and rotation when suspended by the tail were observed after 8 week post-irradiation. Moreover, decreases in histological and behavioral changes were found in the most rats that were injected with the free radical scavenger.

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In order to appear the effects of radiations and radioactive materials, we studied the molecular response to radiation and radioactive materials in yeast cells by using yeast DNA microarray. The yeast cells Saccharomyces cerevisiae cultured in YPD medium (OD=1.0) were irradiated by γ ray (10Gy, 50Gy), X ray (50Gy), heavy-ion radiation (25Gy), fast neutron (3.3, 9.2, 20.3Gy) and thermal neutron+γ ray (3.9, 7.0, 24.5Gy), respectively. In order to examine the effect of radioactive materials, yeast cells were exposed to thorium nitrate (5 mM) for 2h. mRNA was extracted from those samples and analyzed by yeast DNA microarray. Although genes (ex. HUG1, GTT1, DDR2) related to DNA damage and oxidative stress were induced by all irradiation treatment, we could observe characteristics inductions by each treatment. For example, genes encode heat shock stresses and ubiquitins were significantly induced in yeast cells which were treated with thermal neutron + γ ray. Genes related to proteins which were located in mitochondria (FMP) and genes related to transposon were detected with fast neutron. We compared genome-wide expression profiles of these radiation stresses using hierarchical clustering. Expression profiles of γ ray, X ray, heavy-ion radiation, fast neutron and thermal neutron + γ ray produced the same cluster, though those for thorium gained independent. On the basis of these results, it may suggest that yeast cells have characteristic response depending on the kind of radiation ray and radioactive materials.

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With the completion of sequencing of the human and mouse genomes, systematic analyses to elucidate the gene functions are getting to be more important.
OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we succeeded to isolate a novel mutant resistant to diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) from the gene trap insertional mutants library of Chinese hamster ovary (CHO) cells. In this mutant, the OVCA1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin resistant-phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2 (EF-2), the target of bacterial ADP-ribosylating toxins.
The finding that OVCA1 is involved in the diphthamide biosynthesis will shed light for the further understanding of the molecular mechanisms underlying the tumorigenesis in the defect of OVCA1 and the physiological role of diphthamide. Furthermore, a functional genetic approach utilizing the library of random gene-trap mutants of CHO cells should become a useful strategy to identify the genes responsible for specific phenotypes.

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This study was undertaken to optimize the irradiation method of heavy ions to marine seaweeds and to analyze the irradiation effects on Asakusanori (Porphyra tenera) that is the most commercially important seaweed. Both diploid phase (sporophytic filaments) and haploid phase (gametophytic blades) of P. tenera were used. The respective tissues were packed into 4-well shares and 4-well chamber slides for quadricate experiments and then subjected to irradiation with He (150 MeV/u), Ar (500 MeV/u), Ne (400 MeV/u) and C (290 MeV/u) ions between 12.5 and 100 Gy at the HIMAC facility of NIRS. This preceding study verified that both phases showed low sensitivity toward irradiation of He ion with 100 Gy but high sensitivity toward that of C ion even with 12.5 Gy. The same tendency occurred by irradiation of He and C ions was reported from terrestrial plants such as soba seeds, rice, and rose axillary buds. The irradiation effects of He and C ions with a maximum dose of 200 Gy on the growth and maturation of P. tenera gametophytic blades were analyzed. Irradiation of the blades with 50 Gy of He ion beam obviously suppressed their aging, and that with 50 Gy of C ion beam accelerated their production of carpospores and further development to conchocelis. These results indicate that both the optimum conditions for irradiation with He and C ions are found to be a dose of 50 Gy to induce a wide range of mutagenesis in P. tenera gametophytic blades and that marine seaweeds could be highly resistant to irradiation with heavy ions rather than terrestrial plants.

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The methylation of cytosine in the genome is quite important for epigeneticgene-expression regulation. Drastic changes during early development havebeen observed and its critical role is also clear. The "in printing"phenomenon is mediated by the methylation of the genome. Furthermore,methylation at specific sites is responsible for certain diseases.Methylation in the genome is very interesting and important for all lifesciences, including radiobiology.Here we developed a new tool for detecting a difference in methylationbetween various genomes. Using methylation-sensitive and -insensitiverestriction enzymes and HiCEP technology, we achieved extraordinarily highcoverage, high sensitivity analysis of methylation in the genome. Inaddition, isolation of methylation sites after analysis is significantlyeasier than with pre-existing techniques like 2D electrophoresis.In our present procedure, approximately 10,000 methylation sites can becovered in one analysis. More than three analyses can be carried outsimultaneously, enabling us to analyze more than 30,000 sites in thegenome. Isolation and sequencing showed that more that 50% of the sitesdetected were located within the gene region registered in the publicdatabase and the rest were outside the gene regions. We are now trying toidentify novel transcripts in the sites that were mapped outside of thegene regions.

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A search for ATM -related transcripts in the dbEST detected a transcript (AA41225) oriented oppositely of the ATM gene, which includes a part of ATM (exon 53 and flanking introns, intron 57) and a partial sequence 20 kb distant from the ATM 's 3' end. A putative ATM -overlapping gene reflected in AA41225 was further characterized in this study. The gene, localizing between ATM and KDELC2 (KDEL(Lys-Asp-Glu-Leu) containing 2), had at least 9 exons outside the ATM locus. The AA41225 was supposed to be a variant arising from alternative splicing, which has joined exon 7 and a new 73-bp exon within the ATM intron 57 by skipping two exons. There found several variants with different 5' splice sites, whereas the 3' splice site within the ATM intron 57 was commonly shared. Quantitative PCR analysis of human multiple-tissue RNA panel has revealed this transcript is specifically expressed in testis, while the amount of ATM -containing variant is somewhat lower than the counterpart without ATM sequence. The gene expression was very low in primary fibroblasts as well as in a panel of 24 human tumor cell lines. The elevated expression was also seen in mouse testis, whereas the ATM -containing variant was not detected. This suggests the variant may have resulted from recent sequence alterations in the 3' region of this gene within the primate lineage.

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Epidemiological studies on atomic bomb survivors exposed in utero have revealed that radiation, mostly gamma-rays, causes abnormalities of brain development. Several researchers have reported that prenatal irradiation of mouse of low-LET radiation also induces developmental anomaly of brain. However, the effects of prenatal exposure to neutron on brain development have been little understood. In the present study, we investigated the effects of neutron exposure in utero on the brain development and on the behavior pattern of mice at adult stage. B6C3F1 mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.1, 0.2, 0.5 Gy) or Cs-137 gamma-rays (0.8, 1.5 Gy) on gestation day 13.5. At 14 months of age, female offsprings were tested for their spontaneous movement. The brains were removed, weighed, and then were fixed for histopathology. The brain weights decreased in dose-dependent manner in both types of radiation, and the effect was significant for 1.5 Gy of gamma-rays and more than 0.2 Gy of neutrons. Hypoplasia of cerebral cortex was observed for both of the highest dose groups. The spontaneous movement during dark period evidently increased in the mice exposed to more than 0.1 Gy of neutrons and more than 0.5 Gy of gamma-rays. The dose of neutron required for neurobehavior change was nearly 10 times smaller than that of gamma-rays. These results indicate that change of spontaneous movement might be an index for radiation effects more sensitive than brain weight or histopathological changes.

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NBS1 is essential for the repair of radiation induced DNA double-strand breaks (DSBs) in yeast and higher vertebrate animal. We used human cultured non-small cell lung cancer cells, wild-type p53 cells (H1299/wtp53) and mutated p53 cells (H1299/mp53) having identical genetic background except for p53 gene. When a DNA cassette expressing small interference RNA (siRNA) targeted for the NBS1 gene was transfected into these cells by liposome, we observed the suppression of the constitutive expression of NBS1 protein and the suppression of phosphorylated NBS1 protein accumulation after X-ray irradiation. We showed that the radiation sensitivities of wtp53 and mp53 cells were enhanced by NBS1-siRNA. In addition, the radiation-induced cellular survival signaling pathway via NF-κB and XIAP (NF-κB-regulated inhibitory factor of apoptosis) was suppressed by NBS1-siRNA. The induced XIAP expression was strongly suppressed by NBS1-siRNA in mp53 cells as compared with wtp53 cells. Furthermore, we found that NBS1 and phospho-NF-κB were not accumulated after X-ray irradiation in radio-sensitive Gmtert cells (established from a Nijmegen breakage syndrome patient) bearing mutated NBS1 but in 82-6 normal skin fibroblast cells bearing wild-type NBS1. These results suggest that NBS1 mediates to not only to repair of radiation induced DSBs but also radiation-induced cellular survival signaling pathway via NF-κB.

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Whether or not cancer cells are resistant or sensitive to radiation is an important consideration in radiotherapy. It has been widely reported that cells with mutant p53 are more resistant to radiation. On the other hand, there have been reports of cells with mutant p53 that are sensitive to radiation. The positions of mutation were not discussed in these reports. We have constructed an in vitro system to examine how p53 mutants affect radiation sensitivity. We transfected sixteen kinds of mutant p53 and a wild p53 by LacSwitch vector system to human osteosarcoma cells, Saos-2, which are devoid of endogenous p53 gene. We got clones of every mutants and wild type p53, and then examined the sensitivity of the clones to gamma irradiation of 6 Gy. Five mutations of p53 were resistant to radiation compared with wild type p53 cells. Hot spots mutants belonged to this category. Four mutations of p53 were sensitive to radiation like as wild type p53 cells. Seven mutations of p53 showed medium sensitivity to radiation. As the positions of p53 mutations are spread widely throughout the gene, and the radiation sensitivity of cells depends on the position of p53 mutation, it seems to be important to check the mutation position of p53 in tumor cells for predictive assays for radiotherapy.

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Purpose: Some cervical cancers are reported to be resistant to Radiation therapy. We have studied molecular chaperons as intercellular molecules that may get involved in acquisition of radioresistance. This study examined the involvement of GRP94 in radioresistance in human cervical cancer cells.
Methods: Seven human cervical carcinoma cell lines (HeLa, SKG-I, SKG-IIIb, QG-U, Caski, SiHa and C33A) were examined for cellular levels of the GRP94 protein by western blotting analysis. Sensitivity of these cell lines to X-ray irradiation was estimated by a colony survival assay. The suppression of GRP94 expression was performed using specific small-interfering RNA (siRNA) in HeLa and Caski cells.
Results and discussion: HeLa and QG-U cells with high levels of GRP94 exhibited resistance to X-ray cell killing. In contrast, SKG-IIIb, SiHa and C33A cells with low levels of GRP94 exhibited high susceptibility to X-ray. In HeLa cells, the resistance diminished when GRP94 levels were reduced by specific siRNA transfection. However, a reduction in GRP94 amounts did not result in X-ray sensitization in Caski cells, which expressed low levels of GRP94 but showed X-ray resistance. These results suggest that high cellular contents of GRP94 protein is one of the molecular mechanisms enabling resistance of cervical cancer cells to radiation, and that GRP94 siRNA might be useful as a tool of tumor-specific gene therapy in combination with radiation in cervical cancer.

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In order to carry out a suitable radiotherapy, it is important to prevent the outbreak of radioresistant cells. To understand the mechanism of radioresistance, radioresistant cell lines are needed, however, the establishment of cell lines with stable radioresistance has not yet reported. Therefore, we tried to establish radioresistant cell lines with the same genetic background. We exposed cells to medium dose of X-rays every day more than 3 years and obtained 3 independent derivative cell lines. No difference was observed with regard to radiosensitivity determined by clonogenic assay or rejoining kinetics of double strand breaks determined by commet assay among those cell lines. However, every 5th-day exposure to 5 Gy of X-rays revealed that 1 cell line acquired significant profile of radioresistnce. We need refine clonogenic assay or establish a novel method to determine radioresistance in vitro. We conclude that our cell lines are a key to understand the mechanisms of radioresistance and biological relevance of exposures to long-term radiation.

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To characterize gene expression profiles of neonatal human dermal fibroblast (NHDF) cells treated with X-ray or carbon-ion, we performed a genome-wide survey of the temporal transcriptional response of the cells using oligonucleotide microarrays. Total RNA was purified from NHDF cells at 1, 3, 6, 12, 18 and 24 hr after exposure to 0.1, 2 and 5 Gy. Based on array data derived from the X-ray and carbon-ion irradiations, X-ray: 977; carbon-ion: 966 genes with statistically significant differences in exposure dosage and time were isolated by ANOVA. Genes with expression changes of more than 1.5 fold were further analyzed, and their characteristic beam-specific profiles were respectively derived. Interestingly, majority of these genes were down regulated, and changes in the total expression level were higher with carbon-ion irradiation, with the time factor exerting a greater effect on gene expression variations than dosage. Another N-way ANOVA analysis was performed to select 510 genes, and further selection was made to find 70 genes that showed radiation species-dependent gene expression change (fold change>1.25). The genes were then categorized by the k-mean clustering method into 4 clusters; where they were found to display special features with regard to respective cell cycles, cell deaths, responses to stress and metabolisms. Identification and characterization of X-ray- or carbon ion-specific genes may facilitate an understanding of the molecular response mechanisms in the irradiated cells.

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We previously reported that the apoptosis index in jejunal crypt cells was significantly greater in spontaneously hypertensive rats than in Wistar-Kyoto rats after X irradiation and that administration of reserpine to induce sympathetic dysfunction of spontaneously hypertensive rats or Wistar-Kyoto rats resulted in a significant suppression of apoptosis. We hypothesized that hyperfunction of the sympathetic nervous system is involved producing the high susceptibility to radiation-induced apoptosis of the jejunal crypt cells. To clarify this hypothesis, we examined the effect of norepinephrine (NE) on cell survival using the clonogenic assay and apoptosis after X-ray irradiation in rat ileal epithelial cells (IEC-18).
NE treatment in rat ileal epithelial cells resulted in the decrease of cell survival in a dose dependent manner (>1µM). A combination of NE 1µM and radiation decreased surviving fraction at 8 Gy from 24.1% to 6.4 % significantly. NE concentration 1µM resulted in increase the apoptotic rate from 3.5 % to 6.0 % 6 h after 8 Gy irradiation (p<0.05). The NE induced cell sensitivity to radiation was attenuated by alpha1-adrenergic antagonist (prazosin) but was not affected by beta-adrenergic antagonist (propranolol).
Our study suggested that NE enhanced sensitivity to radiation in rat epithelial cells and that may be caused through alpha adrenergic receptor.

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The purpose of this study is to evaluate the relationship between the expression of cytokines and the incidences of metastases and local recurrences in patients with heavy-ion radiotherapy.Serum levels of cytokines such as Il-6¸Il-11 and VEGF¸VEGF-C¸HGF were measured by ELISA analysis in 41 patients with locally recurrent rectal cancer and 39 patients with pancreas cancer.In patients with locally recurrent rectal cancer¸no correlation was found between S–VEGF and S–CEA or CA19–9.No significant correlation was found between S–VEGF before and after heavy–ion particle therapy·The VEGF levels of patients with distant metastasis after therapy(102.4±23.8pg⁄ml) were significantly higher than in patients without metastasis(27.9±4.7pg⁄ml)Similarly¸The HGF levels of patients with distant matastasis (0.461±0.384ng⁄ml) were significantly higher than in patients without metastasis(0.162±0.037ng⁄ml).There was no correlation between VEGF and distant metastasis. However, the VEGF–C levels of patients with lymph node metastasis after therapy(625.1±70.8pg⁄ml) were significantly higher than in patients without metastasis(584.7 ±97.7pg⁄ml). Il–11 was decreased in the patients with severe gastrointestinal damages. These results suggest thet the levels of these cytokines have predictive value for treatment response and outcome of patients with rectal cancer or pancreas cancer.

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NBS1 is well known to play an important role in DNA repair (homologous recombination repair and non-homologous end-joining repair). We aim the radio-sensitization in human cultured tangue cancer cells bearing different p53 gene status of mutation type (m)p53 gene and the wild-type (wt)p53 gene by siRNA targeted for NBS1 through inhibition of the gene expression. The efficiency of transfection of DNA cassette expressing NBS1-siRNA is about 80% in these cell lines. The transfection of the DNA cassette inhibited specifically expression of NBS1 and enhanced radiation sensitivity in these cell lines p53-independently. These results suggest that siRNA targeted for NBS1 is possibly effective for radiation therapy in not only wtp53, but also mp53 cancer cells.

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In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (<1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. We have previously shown that the p21 gene promoter transduced by recombinant adeno-associated virus (rAAV) vectors was highly radiation-responsive. Here, we examine if a suicide gene HSVtk, inducible by a low dose radiation through the p21 gene promoter, enhances sensitivity to radiation of the cells. MCF-7 cells were transduced with rAAV-PtkS, which contains the HSVtk gene linked to the p21 gene promoter. The transduced cells were treated with various concentrations of GCV followed by irradiation with 1 Gy of X rays twice a day with an interval of 5 h for the 5 consecutive days. It was observed that transduction of rAAV-PtkS significantly sensitized cells to X rays. Therefore the p21 gene promoter in combination with a rAAV vector is considered to be potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

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The purpose of this study is to evaluate acute and late skin damages following heavy-ion particle therapy(HRT) by Mexameter(reflectance spectrophotometer) and Cutemeter(viscoelasticmeter). We measured erythema index and melanin index by Mexameter, skin elasticity, distension ability and retraction ability by Cutemeter. We therefore analyzed the correlation between these indexes and DSH(Dose Surface Histogram) calculated using HIPLAN software. We examined 31 patients with locally recurrent rectal cancer. HRT dose ranged from 67.2 to 73.6GyE at 16 fractions with treatment fieldErythema and melanin index increased with time and were highly correlated with elapsed treatment days. Maximum index of erythema and melanin are correlated to S20(skin area irradiated in excess of 20GyE) and S30 (30GyE) respectively. Index of elasticity, distension ability and retraction ability decreased with time after treatment. They reached minimum indexes at 6 months after therapy. Distension ability and retraction ability of skin decreased rapidly compared to skin elasticity. These minimum indexes are related to S20.It was suggested that these indexes were useful for showing skin reactions induced by heavy-ion particle therapy. The values derived from DSH may be useful in comparing competing treatment plans; S20 and S30 were found to be a satisfactory candidate for use as a predictor of acute skin reactions.

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