Rev1, the number of the Y-family DNA polymerase, has deoxycytidyl transferase activity that incorporates deoxycytidine opposite an abasic site. Rev1 plays the central role on the translesion DNA synthesis known as error-prone DNA repair. However, whether Rev1 protein has the precise role in tumor formation in vivo is still unclear. To address this issue, we have generated metallothionein promoter-Rev1 (MT-Rev1) transgenic mice that continuously express a Rev1 transgene in multiple tissues.
Six weeks old MT-Rev1 and wild-type mice were administered repeated ip injection of N-methyl-N-nitrosourea (MNU) and were investigated whether Rev1 overexpression could accelerate the appearance of tumors.
Consequently, the latent period of thymic lymphoma appearance in MNU-treated MT-Rev1 female mice was markedly shorter than that in wild-type female mice. In male, MNU-treated MT-Rev1 mice significantly enhanced the intestinal tumor development in comparison with MNU-treated wild-type male. The number of intestinal tumor was significantly higher in MNU-treated MT-Rev1 male mice than in wild-type male mice.
These findings demonstrate that expression of Rev1 may be associated with induction of tumor in mice.

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Whole-body γ-irradiation to mice causes thymic atrophy and depletion of bone marrow cells, followed by a supply in an improper manner of cells from bone marrow to the thymus. The atrophic state of thymus continues at least to 100 days after and about two thirds of the thymuses develop lymphomas until 300 days after, some remaining apparently unchanged. Thus, those atrophic thymuses at early days may well found a population of proliferating and precancerous cells. Indeed, it was reported that pre-lymphoma cells develop in atrophic thymus within two weeks after irradiation. We examined various atrophic thymuses to characterize pre-lymphoma cells. The cell number was decreased to approximately one tenth and the proportion of large proliferating thymocytes tended to increase at thymuses at 35 and 100 days after irradiation, and this tendency became evident with time after irradiation. PCR analysis for VDJ recombination revealed the existence of clonally growing thymocytes in thymuses at as early as 35 days after. Interestingly, some thymuses exhibited genetic changes such as allelic loss at Bcl11b, indicating the start of clonal expansion of pre-lymphoma cells at a very early stage. On the other hand, BrdU incorporation was decreased with time after irradiation. Taken together, the clonally growing large thymocytes may be pre-lymphoma cells but show hindrance in cell-cycle progression. Therefore, abrogation of this hindrance through additional genetic and/or epigenetic changes is probably a prerequisite for lymphoma development.

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High dose-rate radiation is known to induce acute myeloid leukemia (AML) in human and mice. Recent reports suggest that cancer stem cells initiate and maintain cancer tissue. In this study, we analyzed cell-surface antigen of murine radiation-induced AMLs, to identify the initiation and differentiation stages of AMLs. Eight AMLs were induced in male C3H/He Nrs mice by 3Gy of gamma-ray at a dose rate of 1.0 Gy/min. Spleen cells from each mouse with AML were injected into syngeneic mice. Bone marrows and spleens of recipient mice were analyzed for the cell-surface antigens by flowcytometry. The analysis of initiation and differentiation stage revealed that 4 out of 8 AMLs had an increased cell-population of common myeloid progenitor (CMP)-like cells, and the remaining 4 AMLs were rich in hematopoietic stem cell (HSC)-like cells. All AMLs had similar cellular composites both in the bone marrow and the spleen. Partial losses of chromosome 2 were detected in 2 of 4 CMP-like AMLs by array CGH analysis. Furthermore, we transplanted each of HSC-, CMP- and common lymphoid progenitor (CLP)-like cells separated from a CMP-like AMLs into syngeneic mice. It was shown that the development of AMLs from transplanted CLP-like cells was suppressed or delayed compared to those from HSC- or CMP-like cells. The results indicate that murine radiation-induced AMLs with the partial loss of chromosome 2 were initiated from HSC or CMP, transforming into AML stem cells. This work was supported by a grant from Aomori Prefecture, Ja

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Radiation-induced mouse thymic lymphoma is a useful model for studying not only the mechanism of radiation carcinogenesis but also genetic susceptibility to tumor development. Using array-comparative genomic hybridization, we analyzed genome-wide DNA copy number changes in radiation-induced thymic lymphomas developed in susceptible C57BL/6, resistant C3H and the relatively susceptible hybrids C3B6F1 and B6C3F1. Besides aberrations at known relevant genetic loci including Ikaros and Bcl11b and trisomy of chromosome 15, we newly identified strain-dependent genomic imbalances on chromosomes 5, 10 and 16, and strain-independent trisomy of chromosome 14 as frequent aberrations. In addition, biallelic rearrangements at Tcrb were more frequently detected in tumors of C57BL/6 than C3H, suggesting aberrant V(D)J recombination and a possible link with tumor susceptibility. The frequency and spectrum of these copy-number changes in lymphomas of C3B6F1 and B6C3F1 were similar to that of C57BL/6. Furthermore, in combination with loss of heterozygosity analysis of F1 tumors, it was suggested that allelic losses at Ikaros and Bcl11b were primarily caused by multilocus deletion, whereas those at Cdkn2 and Pten loci were mainly due to uniparental disomy. These findings provide important clues to both the mechanisms for accumulation of aberrations during radiation-induced lymphomagenesis and different susceptibility among mouse strains. This study was supported by a grant from Aomori Prefecture, Japan.

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Recently, a lot of experimental model organisms have been developed. They have the advantage that genetics can be used full. Medaka is most suitable because both of classical and modern molecular genetics are available. Recently a general reverse genetics method was reported, which can identify mutations in genes that are known only by their sequence. The method, called TILLING (Targeting Induced Local Lesions IN Genome), includes random mutagenesis, followed by screening for induced mutations in target genes at the genomic DNA level. Adult male Medaka were mutagenized with ENU and then outcrossed with female to generate F1 progeny for the library. We established about 5,700 ENU-mutagenized F1 male fishes. To construct a library, genomic DNA and testis samples were isolated and cryopreserved from each F1 fish. And we established screening methods. Screening revealed that the average per-base mutation frequency was 1 in 300kbp recently. The thing indicated that can be used enough for screening, and some nonsense mutants have already been obtained. We have introduces the outline of the entire screening including detect of the mutant.

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We have studied combined effect of X-rays and N-ethyl-N-nitrosurea (ENU) on the development of thymic lymphoma (TL) using B6C3Fl mice. Our previous results indicated that the induction of TL by ENU was suppressed by prior irradiation of low dose X-rays (0.2 Gy for 4 consecutive weeks), while high dose irradiation (1 Gy for 4 consecutive weeks) showed synergistic effect. This was coincident with the reduced and enhanced frequency of ENU-induced gpt mutations by prior exposure to low and high dose radiation, respectively. To provide further molecular clue of combined effect, we examined deletion mutations (Spi^-) of thymic cells.
[Material and methods]
Four-weeks-old gpt-delta mice were exposed weekly to whole body X irradiation at 0.2 or I Gy for 4 consecutive weeks. After irradiation, mice were given ENU at concentration of 200 ppm by free-choice drinking for 4 weeks. After 4 weeks of the end of ENU treatment, genome DNA was prepared from the thymus for Spi^- assay, which can detect deletions of red/gam gene.
[Result]
We found that net mutant frequency varied from I to 7 x 10^-6. Mutations occurring in gam gene were predominantly one base deletion in run sequence in control and X-irradiated groups. In ENU- and combined-exposure groups, however, base substitution mutations were also developed. There was little difference in occurrence of large deletion mutation among groups (< 2 folds). These results suggested that, unlike gpt mutation, occurrence of deletion mutations were not affected by pre-irradiation. Taken together, it is suggested that the development of TL by combined exposure was mainly caused by increase or decrease in base substitution, which is dose-dependent.

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Helicases are ubiquitous enzymes that can transiently catalyze the unwinding of nucleic acid duplexes using NTP hydrolysis as the source of energy. They play vital roles in nearly all DNA metabolic processes, including DNA replication, recombination and repair. The defects in some specific DNA helicases can cause genetic diseases, and such helicases have been shown to play roles in protecting cells from DNA damaging agents.
The Pif1 subfamily is a 5' to 3' DNA helicase, which belongs to SF1 superfamily, is found to be conserved from yeast to mammals, suggesting important function for genomic maintenance. In order to elucidate the function of human Pif1 helicase, we cloned hPif1 helicase from HeLa cDNA library. The PIF1 cDNA encoded a 69 kDa protein consisting of 641 amino acid residues. The PIF1 gene consisted of 13 exons located on chromosome 15q22. RT-PCR using commercially available muti-tissue cDNA panels revealed that hPIF1 was ubiquitously expressed in all the tested 16 tissues, higher in the thymus, spleen, testis, and very low in the pancreas, prostate, peripheral leukocytes. For further analysis of the gene product, we purified the full-length recombinant proteins from over produced E. coli cells. We demonstrated that the recombinant protein has a DNA-dependent ATPase activity, and also has 5' to 3' helicase activity, which is much stimulated by fork-structured substrates.

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We plan to develop new assay systems that use GFP for mutant detection. The principle of the approach is to make the cells fluorescent when mutation occurred at specified gene loci in the genome. One example is to introduce cells co-expression of two vectors, one produces GFP constitutively and the other produces repressor protein to silence the expression of GFP gene. In this system, any kinds of forward mutation in the repressor gene allow GFP gene transcription from null state, which should make the mutant cells easy to be recognized by green fluorescence. Another example is to make partial duplications of endogenous gene, in which the distal segment of the duplicate has a GFP gene tag. In this system, reversion from the duplicate makes the mutant cells fluorescent. Toward the generation of mouse model systems, we have been evaluating the systems in cultured cells and measuring spontaneous and radiation-induced mutants by flow cytometry.

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The ion beams were widely used as the mutagen for plants and also used for cancer therapy. However, precise effect of ion beams to DNA molecules is not yet elucidate. The molecular mechanism of the mutagenesis caused by carbon ion beams was studied. The mutation sites caused by ion beams were determined and the mutation spectrums were compared with those induced by gamma ray.
S. cerevisiae strains used in this study are S288C (RAD⁺), rad52 and ogg1. RAD52p is a component the homologous recombination repair pathway. OGG1p is a DNA glycosylase / AP lyase that excises guanine lesions such as 8-oxoguanine (8-oxoG).
The yeast strains were irradiated with carbon ions (220 MeV) with the dose 10 to 100 Gy, and LET is 107 keV/μm. Carbon ion beams were generated from AVF cyclotron in JAEA. The mutation sites of ura3 mutants were determined by DNA sequencing. The URA3 regions of mutants were sequenced. The carbon ion induced more transversions than transitions, deletions or insertions. Moreover, the fact that GC to TA transversions largely observed suggests that the mutations by ion beams resulted from oxidative damage such as formation of 8-oxodG mainly, and the remarkable feature of yeast mutations induced by carbon ions was that the mutations sites were localized near the linker regions of nucleosomes.

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In recent years, it was reported that the frequency of mutation and chromosomal aberrations are significantly elevated among progeny of X-ray survivors in comparison with un-irradiated cells. This phenomenon is called "genomic instability" and it may be an important step of carcinogenesis. It is thought that DNA double strand breaks are essential in order to induce genomic instability, but the mechanism of maintaining instability and expressing delayed effect is still unknown.
Because chromosomal deletion occurs as a result of DNA double strand break repair, we predict that genomic instability is induced by the large-scale structural change which happens after irradiation. To make sure this assumption, we analyzed the size of gene deletion and the chromosomal aberration frequency after irradiation.
In the experiment, we irradiated normal human diploid cells with X-rays and isolated HPRT mutated-clones. Then, we analyzed the abnormal chromosome by using WCP FISH. In addition, we examined presence of each exons of HPRT gene and STS markers to assess the size of gene deletion. In the result, we found out that occurrence rate of chromosomal aberrations in clones with a large deletion (>0.5Mb) rises significantly. Furthermore, we isolated secondary and tertiary clones from primary clones with a large deletion (>0.5Mb), and analyzed occurrence rate of chromosomal aberrations.
As a result, translocation is dominant aberration in primary clones. In secondary and tertiary clones, translocation frequency declined, and dicentric is dominant aberration. Clones with the large deletion induce break of the chromosome and telomeric instability. We also found out that telomeric instability is maintained over a protracted period of time after irradiation.

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Mouse genome contains thousands of copies of retrotransposon, intracisternal A-particle (IAP) DNA element, that is constructed from gag-pol gene sandwiched betweem two LTR sequence. The transcripts of IAP can be converted to cDNA and then integrated into the mouse genome. The IAP-mediated retrotransposition may occur in any of the mouse cells. We have previously found that genomic rearrangement by the IAP-mediated retrotransposition frequently occurs in acute myeloid leukamia cells of C3H/He mouse. To study the mechanism of the IAP-mediated retrotransposition in the presence of large amonts of structurally-related nucleic acids, we established a reporter gene system for reverse transcription.
We constructed transgenes to be designed to express IAP RNA with unique nucleotide sequence markers as a reporter gene for reverse transcription. Series of intermediate cDNA molecule during retrotransposition process such as a tRNA-Phe-primed cDNA molecules were idenitifed in the stable transfectant of mouse RAW264.7 cells with the transgenes. Quantitative methods to measure the faint amount of these cDNAs based on real-time (RT)-PCR were established. The amount of trangene cDNAs increased following exporeuse of X-ray in the irradiation-dose dependent manner. This suggests that radiation stimulates retroviral reverse transcription process in the mammalian cells.

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C3H/He inbred mice frequently generate myeloid leukemia after sublethal doses of ionizing radiation. We have previously found that the unique genomic integration of cDNA of retrotransposon, intracisternal A-particle (IAP), reversely transcribed from IAP RNA is frequently detected in radiation-induced myeloid leukemia cells in the C3H/He mice. However, the molecular mechanims of the retrotransposition of repetitive sequence of mouse genome have not been understood. To investigate synthesis of IAP cDNA from IAP RNA in C3H/He mouse, our original methodology to measure reverse transcription in cell lines was expanded to the transgenic mouse systems in this study. Transgenes designed to express IAP RNA constitutively with unique oligonucleotides as molecular probes were constructed. By the direct injection of the 12.5-14.1 kb transgenes into embryos of C3H/He inbred mouse, we have succesfully generated 19 lines of transgenic mice. Finally, 7 mouse lines with different homogeneous allele of the transgene were established. Occurrence of reverse transcription were confirmed by the detection of unique cDNA molecules derived from transgene RNA in the cells of the transgenic mice. The faint amounts of cDNAs in the various cells in the mice were measured by the quantitative real-time PCR. Higher levels of the cDNAs were observed in radiation sensitive cells including myeloid, intestine and germline cells. The results showed that the transgenic mice is useful to study molecular process of reverse transcription in the retrotransposition.

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Although radiation-induced genomic instability is well characterized in vitro by the delayed appearance of specific types of genomic damage, (e.g., chromosomal aberrations, gene mutations, etc.), in the progeny of irradiated cells, evidence from in vivo studies has been limited due to lack of reliable bioassays that are capable of detecting delayed genomic alterations induced by irradiation in large cell populations in an objective and straightforward manner. In the present study, using flow cytometry, we quantified the number of micronucleated reticulocytes in peripheral blood of whole-body X-irradiated mice in order to evaluate the radiation sensitivity and the induced genomic instability of the hematopoietic system. An acute effect of radiation dose as small as 0.1 Gy was detectable two days after irradiation, and the radiation dose effect was significantly greater in BALB/c mice than in C57BL/6 mice (p<0.001), i.e., 3.0- (p=0.002) and 2.3-fold (p=0.002) increases in frequencies of micronuclei were noted in the two groups of mice, respectively. Even one year after irradiation, mice irradiated with 2.5 Gy of X-rays showed significantly increased frequencies of micronucleated reticulocytes, i.e., 1.6- (p=0.035) and 1.3-fold (p=0.039) increases in BALB/c and C57BL/6 mice, respectively. A significant mouse strain difference in the delayed radiation effect was noted as well (p=0.028). The results indicate that delayed genomic effects of irradiation on the murine hematopoietic system can persist in vivo for prolonged periods, and that there are mouse strain differences in sensitivity to radiation-induced genomic instability.

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Nijmegen syndrome is the genetic disorder, which is characterized by high sensitivity to radiation, chromosome instability and aberrant cell cycle checkpoint. NBS1, the gene responsible for Nijmegen syndrome, forms a protein complex with hMRE11 nuclease and hRAD50, and functions in homologous recombination (HR) repair from DNA double strand breaks, which are elicited by ionizing radiation or other stresses. NBS1 also binds to ATM at the C-terminus and disruption of the interaction fails to recruit ATM to damage sites. Both ATM and NBS1 could have pivotal roles in regulations of cell cycle checkpoints. Therefore, the chromosome instability in NBS cells is considered to be due to defects in both DNA repair and cell cycle checkpoints. Thus, DNA repair and cell cycle checkpoint genes are responsible for genome instability. However, it is suggested that genome instability in tumor is related to centrosome aberration. Centrosome is the complex organelles comprising two microtuble-based centrioles surrounded by a protein matrix (pericentriolar material, PCM) and other structural elements, a key regulator for chromosome separation in mitosis. Proper centorosome duplication and spindle formation are crucial for prevention of chromosomal instability. Therefore, the normal function of centrosome is essential for maintenance of genome stability. Recent studies suggest that DNA repair factors are involved in centrosome function. When we examined the localization of NBS1 and ATM by using both protein antibodies, they showed to be accumulated in centrosomes. Moreover, NBS cells showed defect in centrosome amplification, suggesting an indispensable role of NBS1 in centrosome maintenance. We further discuss this novel role of NBS1 in centrosome maintenance.

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Lymphomas induced by radiation in the cross between BALB/c and STS, a susceptible and a resistant strain for lymphomagenesis, show a wide-range of allelic loss on chromosome 4, where preferential loss of the STS allele occurs. This suggests the existence of tumor susceptibility gene(s) on chromosome 4. In the present study, by the use of C.S congenic strains with STS-derived portions of chromosome 4 on the BALB/c background possible correlation of the regions bearing lymphoma susceptibility genes with LOH was examined. Lymphomas were induced in animals with fractionated X-irradiation according to Kaplan's protocol and examined for LOH of chromosome 4. The order and map positions in centimorgan (cM) for microsatellite markers are as follows: D4Mit39(11)-D4Mit17(31)-D4Mit7(36)-D4Mit86(38)-D4Mit302(43)-D4Mit9(45) -D4Mit31(51), spanning the proximal-middle chromosome 4. Allelic loss was seen in 11 (32%) of 34 (C.S17-31 x BALB/c)F₁ as well as in 10 (29%) of 34 (C.S302-9 x BALB/c)F₁, with preferential loss of the STS allele (10/11 and 9/10). On the other hand, both (C.S39-86 x BALB/c)F₂ and (C.S7-86 x BALB/c)F₂.scarcely showed LOH (<5%). The results suggested that the wide-ranging LOH. was a concomitant event of allelic loss in a small segment bearing a potential tumor susceptibility gene in the middle chromosome 4.

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To elucidate the molecular mechanism in radiation tumorigenesis in mice, we generated a mouse strain by substituting an entire chromosome18 in B6-Min mouse. The consomic-Min mouse strain is highly susceptible to radiation tumorigenesis; mice exposed to 2.0 Gy of X-rays at 2 weeks of age induced 7-fold small intestinal tumors and 29-fold colonic tumors over the unirradiated mice. The enhancement depended on the age at exposure; mice exposed at 7 weeks of age had lost the susceptibility to the radiation tumorigenesis. Radiation-induced colonic tumors exhibited higher frequency of allele loss in the Apc gene compared to the spontaneous tumors, while no such difference was observed for the small intestinal tumors. To clarify the molecular events leading to the allele loss in the Apc gene, we developed an algorithm and parameters, using statistical programming language R, that improve the power and accuracy of LOH detection with SSLP markers. Using the algorithm and parameters, we examined for the presence or absence of LOH throughout the whole chromosome 18. The results showed that the vast majority of the radiation-induced intestinal tumors exhibited intra-chromosomal recombination, while spontaneous tumors showed entire loss of the chromosome 18 without any sign of the recombination.

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Purpose: Inactivation of PTEN has been reported in many human and mouse tumors. DNA methylation in non-small cell lung cancer and DNA mutations in endometrial carcinomas, glioblastomas and Cowden disease are frequently reported. Based on reduced expression and frequent loss of heterozygosity (LOH) at Pten locus, epigenetic inactivation of Pten has been suggested in radiation-induced mouse thymic lymphomas (TLs). In this study, we have examined the epigenetic and genetic alteration of Pten in TLs. Materials and methods: The 4-week-old female B6C3F1 mice were exposed weekly to 2.0Gy whole-body X irradiation for 4 consecutive weeks and monitored daily until moribund, and then autopsied. First, DNA extracted from TLs was typed using microsatellite markers for the identification of LOH. Then, we examined expression of Pten by RT-PCR and pattern of DNA methylation by bisulfite sequencing. Results/Conclusion: Pten is mapped on the 24.5cM region from centromere of mouse chromosome 19. LOH was frequently found (6/23, 26%) at this region. TLs showed reduced expression (12/23, 52%) or transcriptionally silencing of Pten (1/23, 4%). However, no aberrant DNA methylation was detected in a CpG island of untranslated Exon 1 and its 5' upstream region. The present results suggest that DNA methylation did not contribute to transcriptional silencing of Pten in TLs. One TL showed aberrant splicing of Pten, which has not been reported so far. In a search for point mutations in the Pten coding region, the RT-PCR products from the others are currently being sequenced.

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The long-lived radical produced by radiation exposure results in the delayed-type point mutation and transformation in vitro. Addition of vitamin C (VC) just after irradiation reduces the frequencies of point mutation and transformation. The data on the role of long-lived radical in radiation carcinogenesis in vivo are insufficient. In this experiment, we investigated an involvement of long-lived radical in radiation-induced mouse lymphomagenesis. We determined the effect of VC on incidence of thymic lymphoma (TL) and the mutation pattern of cancer-related genes in them.
Four-weeks old female B6C3F1 mice were exposed to X-rays (1.4 Gy per week) for 4 consecutive weeks. VC derivative sodium-L-ascobyl-2-phosohate, which shows long half-life in vivo, was used as anti-radical agent (100 mg / kg). Three experimental groups were set, (i) irradiation alone (X-rays), (ii) irradiation followed by VC just after X-rays (X+VC) and (iii) (X+VC) with additional VC treatment for subsequent three months (X+VC continue). We analyzed the gene expression, the point mutation, and the protein expression of tumor suppressor gene Ikaros to which mutation pattern was clarified in radiation-induced TL.
Latent period was shortened in (X +VC) group, but extended in (X+VC continue) group, compared to X-rays group. Interestingly, the latter group lacked point mutation, but not aberrant splicing, of Ikaros. These results suggest an involvement of long-lived radical in radiation-induced lymphomagenesis.

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Suppression of thymic lymphoma induction by low-dose (0.075 Gy) pre-irradiation in C57BL mice irradiated with 4 consecutive doses of 1.6 Gy was already reported. Also, it is known that lymphoid tumors develop in nonirradiated syngeneic thymic grafts implanted into thymectomized, irradiated mice. This indirect effect of radiation on the induction of lymphomas indicates one of the mechanisms for radiation carcinogenesis. The indirect effect might be caused by hypoplasia of grafted thymus, which might increase genetic and epigenetic plasticity during forced proliferation of cells. Here we studied the effect of low-dose pre-irradiation on carcinogenesis in the thymus graft system. As group A, 51 B10.Thy1.2 mice were thymectomized and irradiated with 4 consecutive doses of1.6 Gy at 7-days intervals from 5 wks of age, and as group B, 51 mice were pre-exposed to low-dose irradiation of 0.075 Gy 6 hr before every 1.6 Gy irradiation. Thymuses from new born C57BL/10.Thy1.1 mice were implanted subcutaneously in B10.Thy1.2 mice within 1hr after the final irradiation. The origins of the tumor were assayed by flow cytometry after treatment with anti-Thy1.1 or anti-Thy1.2 antibody. In both groups, tumor incidence of graft origin was 31%, and the mean latencies of groups A and B were 159 and 140 days, respectively(p=0.08). Although the difference was not significant, a delay in lymphoma development suggest that pre-irradiation has a little effect on the suppression of indirect action of radiation on lymphomagenesis.

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Radiation-induced mouse thymic lymphomas (TL) show over-expression of IL-9R and dysregulation of downstream Jak-Stat signal transduction pathway. However, the mechanism of transcriptional dysregulation of Il9r gene was little known. In this study, we established Il9r-high expressing TL cell line, SKY8699, to examine the promoter activity region and key transcription factor of Il9r. First, the transcription starting site was compared among normal thymus, primary TL and SKY8699 by 5' RACE method. We found new transcription starting sites, which was located between 50bp to 330bp upstream of exon1. The starting site was different between normal thymocytes and TL cells. Next we focused on 2000bp upstream of exon1 in Il9r to search for the promoter by luciferase reporter assay. Seven kinds of promoter DNAs with different length were amplified from normal thymocytes by PCR, and inserted to pGL4.10[luc2] vector in order to make Il9r promoter-luciferase vectors. After transfecting the vector into SKY8699 cell, promoter activity was measured by Dual-Glo Luciferase Assay System. Preliminary data suggest that the promoter was located in this region. Further analysis of this region and key transcription factor that is binding to this Il9r transcription region are being investigated.

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Children are more susceptible to carcinogens than adults. Estrogen and progesterone play important roles in controlling proliferation of adult normal mammary cells and hormone receptor-positive breast cancers. In the prepubertal mammary gland, however, cell proliferation is slow and independent of hormonal control. It is not well understood whether hormones play crucial roles in carcinogenesis initiated by prepubertal carcinogenic exposure. The present study aimed at clarifying the cancer-inducing effect of prepubertal carcinogen exposure and the hormone receptor status of resulting tumors as compared to adulthood exposure. Female Sprague-Dawley rats at 3 and 7 weeks of age (pre- and postpubertal stages, respectively) were either gamma-irradiated (2 Gy) or injected intraperitoneally with 1-methyl-1-nitrosourea (MNU; 20 mg/kg) and sacrificed at 50 weeks of age. After histological examination, estrogen and progesterone receptor statuses of carcinomas were examined immunohistochemically, in which the fraction of immunopositive cells per 1000–2000 cells was assessed. As a result, the incidence of carcinoma after MNU exposure was 39% and 17% in the prepubertal and postpubertal exposure groups, respectively, whereas that after radiation exposure was 28% and 55%, respectively. The fraction of hormone receptor-positive cells was 69–100% in postpubertal exposure groups, whether radiation or MNU, and 69% and 32% in postpubertal MNU and radiation exposure groups, respectively. These results suggest the difference between carcinogenic mechanisms of pre- and postpubertal radiation exposures and imply poor prognosis of breast cancers that would develop in prepubertally irradiated women.

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In recent years, new biological findings have substantially increased our understanding of the effects of physical stimulation (cell adhesion or cell shape, etc.) on the process of cell carcinogenesis. Unlike the experiment in vitro, the carcinogenic process in vivo progresses in the various environmental conditions, however, effects of these environmental conditions, including type of mutation, on tumorigenic process needs further investigation. Here, we are going to introduce the mathematical model of tumor growth based on the morphological study.

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Background: the ATM gene plays a key role in ionizing radiation (IR)-induced DNA damage sensing. Purpose of the study was to assess possible correlation between ATM single nucleotide polymorphisms (SNPs) and papillary thyroid cancer (PTC) of different etiology in children. Methods:rs1801516 G>A (exon 39), rs664677 T>C (intron 22) and rs609429 C>G (intron 48) ATM SNPs were profiled in 85 Caucasian patients with pediatric IR-induced (n=40) and sporadic (n=49) PTCs by PCR/RFLP and direct sequencing. Statistical analysis was done using Fisher's exact test. Results: The shift towards the rare allele-carrying rs1801516 genotypes was found in pediatric IR-induced PTCs compared to sporadic ones (47.5% vs. 24.5%; P=0.03; OR=2.8; 95% CI 1.1-6.9). Unexpectedly, the analysis of rs1801516/rs664677/rs609429 SNP combinations revealed no individuals with homozygous wild-type GG/TT/CC genotype among IR-induced cases. In the sporadic PTC group, the frequency of this genotype was 14.6%. The most common genotype detected in both groups was GG/TC/CG (29.7% vs. 43.8% in IR-induced and sporadic PTCs, respectively; P=0.3; OR=0.5; 95% CI 0.2-1.3). The GA/TC/CG combination showed a tendency to dominate in the IR-induced group compared to sporadic PTC (16.2% vs. 4.2%; P=0.07; OR=4.4; 95% CI 0.8-23.0). Conclusion: ATM rs1801516 harboring the rare allele (mostly the GA) and the GA/TC/CG genotype may contribute to the risk of IR-induced PTC in childhood; however this observation needs to be confirmed using a larger sample size.

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Using electronic spin resonance (ESR), our laboratory found a new type of radical (LLRs) with a long life-time in room temperature cells (T1/2>20h). These may play a more important role of induction of mutation and transformation rather than active, short-lived radicals (H, or OH radicals). It is known that dimethyl sulfoxide (DMSO) and L-ascorbic acid (AsA) scavenge short-lived radicals well. On the other hand, AsA can scavenge Long-Lived radicals (LLRs) by post-irradiation treatment (20 min after X-irradiation). And also post-irradiation treatment by AsA reduced mutation frequency in human cells. On the other hand, DMSO treatment reduced chromosomal aberration and mutation by only pre-irradiation treatment. These findings suggest a possibility that a radical which induce mutation differs from an other radical which induce chromosomal aberration, and the former is LLR which is scavenged by AsA, the latter is short-lived radicals which are scavenged by DMSO. Therefore, we studied the difference of radiation protection effect between AsA and DMSO in human diploid cells, to clarify the origin of radiation induced mutation and carcinogenesis.

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The immortal strand hypothesis (J. Cairns, 1975) proposes that asymmetrically dividing stem cells selectively retain chromosomes that bear the older template DNA strands to avoid accumulation of mutations emerged by replication errors. However, there is lack of evidence to support this hypothesis. Recently, neural stem cells can be isolated and cultured by means of the neurosphere culture. This culture method makes it possible to examine whether the immortal strand hypothesis is correct or not. Therefore, we investigated asymmetrical segregation of chromosomes that bear the older template DNA strands in mouse neural stem cells. Neural stem cells derived from adult and embryo mouse forebrain subventricular zone were isolated and cultured for a week by the neurosphere culture method. Then, neurosphere cells were cultured in the medium containing 5-bromo-2-deoxyuridine (BrdU) for 48 hr to label de novo synthesized DNA. After removal of BrdU, the cells were cultured for an additional week (estimated over 7 cell divisions), fixed and immunostained with anti-BrdU antibodies. Mouse embryo fibroblasts (MEFs) were used for controls. The result revealed that 10% of nuclei derived from both adult and embryo neurosphere cells were BrdU-positive and 3% of them were totally labeled with BrdU over seven cell divisions after removal of BrdU where the random chromosome segregation makes it impossible to detect BrdU positive nuclei. In contrast, only 0.2% of nuclei of MEFs were BrdU-positive. Therefore, the present study demonstrates that neurosphere cells contain subpopulations that selectively retain older template DNA strands, supporting the immortal strand hypothesis.

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We previously found that a significant increase of body weight (BW) in SPF B6C3F1 female mice continuously irradiated with low-dose rate gamma-rays (21 mGy/ 22 h/ day). Here we examined the relationships between the increase of BW and amounts of food intake, drinking waters and feces in irradiated mice kept individually during low-dose rate gamma-irradiation. The following results were obtained; 1) The BW of irradiated mice was significantly heavier than that of non-irradiated mice at 42 weeks of age (33 weeks after the start of continuous irradiation). 2) A strong correlation was observed between the magnitudes of the BW at 42 weeks of age and at 17 to 41 weeks of age. 3) When, based on the BW at 42 weeks of age, irradiated mice (n=18) were divided into heavy (n=9) and light (n=9) weighted mice, BW of the heavy-weighted, irradiated mice was significantly heavier than those of heavy-weighted, non-irradiated mice. 4) No significant relations for the BW-increase in irradiated mice was observed to amounts of food intake and drinking water as well as amount of feces. The amount of feces and a ratio of feces to food intake of irradiated mice were, however, significantly higher than those of non-irradiated mice during 3 to 10 weeks after the start of continuous irradiation. This study was supported by a grant from Aomori Prefecture, Japan.

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While genetic studies have shown that under non-stressed conditions both MDM2 and MDMX are essential to maintain p53 in its latent form, the underlying molecular mechanism of how these inhibitors cooperate and regulate p53 is still unclear. To gain insight into this question, we have generated various chimeric proteins and demonstrated that MDM2 and MDMX can form a complex. In this study, by using a series of chimeric proteins between p53 and MDM2-family, we observed that MDM2/MDMX heterocomplex has the ability to ubiquitinate p53 far more efficiently than MDM2 alone. In vivo, MDM2/MDMX heterocomplexes are the predominant form versus the MDM2 homocomplexs. Disruption of the binding between MDM2 and MDMX resulted in a marked increase in both abundance and activity of p53, emphasizing the functional importance of this heterocomplex in p53 control. These results suggest that the complex formation between Ring finger domains of MDM2-family is essential for E3 ubiqutin ligase activity. It has been shown that MDMX was rapidly degraded in response to DNA damage, a phenomenon which is critical for p53 stabilization and activation. This degradation is mediated by MDM2, which via a distinct mechanism preferentially ubiquitinates MDMX ensuring optimal p53 activation. We propose that the interaction between MDM2 and MDMX represents an important mode of p53 regulation.

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Ionizing radiation (IR) has been shown to activate epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). EGFR activation by IR initiates the Ras/Raf/ERK signaling cascade, stimulates cell proliferation, and leads cells to be resistant to IR. The molecular mechanisms underlying IR-induced activation of EGFR are not clear. We have previously reported that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation. In the present study, we investigated the effects of carbon-beam on activities of ERK1/2, EGFR, and Src as indicated by their tyrosine phosphorylation and effects of AG1478 on them using human breast cancer cell line MDA-MB-468. Exposure of MDA-MB-468 cells to carbon-beam caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. AG1478 inhibited carbon-beam-induced ERK1/2 activation. Carbon-beam did not induced EGFR autophosphorylation at Tyr992, Tyr1045 and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. Src became activated as indicated by phosphorylation at Tyr416. These data suggest that carbon-beam activates ERK1/2 through transactivation of EGFR.

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Rho guanine nucleotide dissociation inhibitors (RhoGDIs) regulate the activity of Rho family GTPases. RhoGDIβ (LyGDI/GDID4/RhoGDI2) has two caspase cleavage sites after Asp19 and Asp55. The resulting cleavage products, ΔN(1-19)RhoGDIβ and ΔN(1-55)RhoGDIβ, are expressed in cells under conditions that activate caspases. ΔN(1-19)RhoGDIβ, which can inhibit GDP dissociation, is implicated in the process of apoptosis, and ΔN(1-55)RhoGDIβ, which lacks the ability to inhibit GDP dissociation, is implicated in the process of anoikis, a form of apoptosis which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix. Here, we found a novel cleavage site of RhoGDIβ in X-irradiated BALB/c 3T3 1-1src cells. This site, which contains the consensus sequence for cleavage by downstream caspases, is likely to be cleaved by an unknown caspase other than caspases-1, -3, -7, -8, and -9 because neither caspases are activated in the irradiated 1-1src cells. Diverse forms of cleaved RhoGDIβ represent a guaranteed system for cell death generated by intracellular signaling network.

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We aimed to delineate the molecular structures and mechanisms for apoptotic cytochrome c (Cytc) release upon radiation DNA damage to Molt-4 cells by applying the apoptosis inhibitors of the Bcl-XL BH4 peptide and a VDAC1 inhibitor ruthenium red (RuR). IR-induced DBSs upregulates transcriptionally BH3-only PUMA and Noxa through the ATM-p53 activation and induced apoptosis effectively. RNAi of p53 and Bax effectively inhibited IR-induced apoptosis. Without inhibitors, the above BH3-only proteins activated Bax/Bak (IP studies) to result in Bax/Bak oligomers and Bax (Bak)/VDAC1-hybrid dimer (HD)/monomer (HM) in the cross-linked mitochondria (MT). However, the BH4 and RuR completely inhibited IR-induced apoptosis without affecting Bax/Bak activation and oligomerization. Remarkably, the two inhibitors suppressed the HD formation and Cytc release, suggesting an unanticipated, more important role of the HD than oligomers in the MT for cellular apoptosis induced by IR-induced signaling axis of ATM/p53/selected Bcl-2s.

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Ionizing radiations induce DNA DSBs to the genome. Cell cycling is then suppressed by the damage responsible checkpoints from which cell-cycle progression are released only after completion of DSB repair. Interestingly however, descendants of the damaged cells are prone to exhibit delayed genomic instability in eukaryotes, suggesting a memory of the damage whose mechanism is poorly understood. We have studied delayed recombination in the fission yeast Schizosaccharomyces pombe to investigate its molecular mechanism. X-irradiation increased recombination frequency of a marker with 200 bp tandem repeats dose dependently from 1.0x10^-2% to ~10x10^-2% at a dose of 500 Gy and the elevated frequency persisted for at least 8-10 cell generations. Production of ROS and other bystander factors were not involved in the delayed recombination. In addition, a single DSB in the genome was found to induce recombination in trans at a site elsewhere in the genome. In parallel to the elevated recombination, Rad22 (a Rad52 homologue) focus persisted in the irradiated cells for 10 generations after irradiation. Comparison of expression profiling by HiCEP revealed that at least 45 transcripts exhibited changes in their expression level along with the delayed recombination and that more than 100 transcripts changed their expression level at least 13 generations. These results indicate that damage is memorized by cell even after completion of repair of damage and that genome-wide change of transcription and activation of recombination machinery operate at the downstream of the memory mechanism.

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Double-stranded DNA breaks (DSBs) induced by irradiation is sensed mainly by the ATM signaling cascade. ATM protein is involved not only in the regulation of DNA repair activities and apoptosis but also in DNA damage cell cycle checkpoints. ATM induces the expression of DNA repair genes and cell cycle-related genes following irradiation immediately. In yeast or mammalian cells, ionizing radiation induces a strong, rapid, and transient transcriptional activation of genes involved in the cell cycle response to or repair of DSBs. It is suggested that basic machinery that repairs DSBs and controls cell cycle progression in plants is similar to that of yeast and mammals. Although the ATM pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle and repair DSBs in response to the loss of genome integrity are unclear.
Arabidopsis thaliana (ecotype Columbia) plants were grown under long day conditions on germination medium. For proton beam irradiation treatments, 5-day-old plantlets were irradiated with 200MeV proton beams. Plant material was harvested and total RNA was extracted according to a time course. The transcript profiles of Arabidopsis thaliana homologues of human DNA repair genes and Arabidopsis thaliana homologues of human cell cycle-related genes following irradiation were determined by real-time RT-PCR.

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Human T-cell leukemia cell line MOLT-4 is highly radiosensitive and shows typical apoptotic cell death after irradiation. p53 is a well-studied transcription factor associated with the cell death. Previous studies by us and others have demonstrated that overexpressing p53 shRNA or dominant negative form of p53 in MOLT-4 cells results in resistance to radiation-induced apoptosis. On the other hand, several researchers have reported a novel apoptotic function of p53, i.e., transcription-independent apoptogenic role of p53 in mitochondria. Moreover, recent studies have shown that codon 72 polymorphic variants of p53 show different sensitivities to apoptosis. Arg72 variant of p53 has more potent apoptosis-inducing activity in mitochondria than Pro72 variant.
In this study, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Immunoblotting analysis of p53 in 10 Gy-gamma-irradiated MOLT-4 and Nalm-6 cells revealed that the radiation-induced p53 in Nalm-6 cell showed mostly same electrophoretic mobility as Pro72 p53 from SaOs-2 transfectant, and MOLT-4 cell's p53 showed different mobility. The altered mobility of MOLT-4 p53 suggested the possibility that the p53 was Arg72 variant. Therfore, we next analyzed p53 exon4 genomic DNA sequences encoding codon 72. As expected, MOLT-4 was homozygous with respect to the allele encoding Arg72. The relative contribution of Arg 72 variant in irradiated MOLT-4 transcription-dependent/independent apoptosis would be examined and presented.

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Heme oxygenase-1 (HO-1) is known as a multiple functional enzyme. Its function is not only to catalyze heme degradation but also to contribute to cytoprotection against damages such as the ionizing radiation, oxidative stresses and the ischemia-reperfusion. We previously reported that several polyphenols such as caffeic acid phenethyl ester (CAPE) from propolis drastically induced HO-1 gene in the structure-dependent manner in mouse monocyte-macrophage cells. To reveal the drastic inductive pathway, the nucleotide sequence that is responsive to CAPE in HO-1 gene was assessed by reporter gene RNA assay. Five different regions of nucleotide sequences corresponding to HO-1 gene locus were cloned from mouse genome using PCR and were connected with a luciferase reporter plasmid. Series of the reporter plasmids were introduced into mouse monocyte-macrophage cells (RAW264.7) by electroporation together with a reference gene as an internal standard . The cells were incubated for 4 hours, treated with CAPE for 4 hours and harvested to isolate RNA. RNAs derived from the exogenously introduced reporter gene and reference gene were quantified by real-time RT-PCR using TaqMan(R) probe. The increase in the reporter gene RNA by CAPE was observed in only one reporter gene that possesses 4050 nucleotide-upstream to the 1st exon. This suggests the CAPE-responsive region is localized in the region spanning from 4050 nucleotide upstream to the 1st exon.

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Escherichia coli cells maintain a strong defense against oxidative stress caused by reactive oxygen species such as superoxide radical, hydrogen peroxide and hydroxyl radical. Responding to the elevated flux of superoxide radicals, the expression of more than 30 proteins becomes elevated over the basal levels in E. coli. We have isolated eight strains of Escherichia coli K12 with superoxide-inducible gene soi::lacZ fusion by using Mud(Ap,lac) phage, which is randomely integrated into the E. coli chromosome. The expression of these soi::lacZ genes depended on the increased level of superoxide and the function of soxRS gene. The soi-9 gene was identified as the ydbK gene, which product was predicted as a putative pyruvate oxidoreductase. The ydbK gene was conserved among bacteria such as Salmonella typhimurium. Among YdbK proteins of these species, the ferredoxin:oxidoreductase motif, ferric ion cluster motif, TPP binding motif were conserved. The deficiency in the function of the ydbK gene enhanced the sensitivity of E. coli cells to the superoxide radical-generating drugs, menadione and methyl viologen. Induction of the ydbK gene is necessary for the protection of cells from superoxide radical, even if E. coli cells have two normal superoxide dismutase, SodA and SodB. The ydbK promoter was cloned, and we constructed ydbK::lacZ reporter plasmid. Cells carrying this plasmid showed enhanced LacZ activity in the response to the high level of superoxide radical, depending on soxS gene function.

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It has been known that murine squamous cell carcinoma cell line "NR-S1" is more radioresistant than murine squamous cell carcinoma cell line "SCCVII". Five monoclonal cell lines those have different phenotypes and characteristics have been obtained.and subclassified into NR-S1a, -b, -c, -d and -e. As a result of colony survival assay after irradiation of 0Gy, 2Gy, 4Gy, 6Gy, 8Gy to these monclonal cell lines, a significant difference in in-vitro radiosensitivity between these cell lines were observed (p<0.001). Xenografts of these cell lines were then established in C3H mice, and tumor growth curves of the xenografts after gamma-irradiation of 0Gy, 30Gy and 50Gy were analyzed. The results showed similar radiosensitivity to the in-vitro radiosensitivity in case of 50Gy irradiation. No significant differences in tumor growth curves were found in the cases of 0Gy and 30Gy. Further, array CGH (comparative genomic hybridization) was performed to compare the genomic variation of these 5 cell lines. From the results of array CGH, the genome of each NR-S1 subtype cell line showed amplification of 8th chromosome, loss of 14th chromosome and other variations compared to that of normal liver cell. Most part of the genome was highly homogeneous but some parts of the genome were different according to cell lines. Further investigation of relationship between the genomic variation and phenotype of cell lines is required.

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[Purpose] To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma, by examining cell survival, gene expression profiles, and cell cycle progression. [Materials and Methods] We exposed 6 melanoma cell lines (C32TG, Colo679, HMV-I, HMV-II, 92-1, and MeWo) to C-ions or X-rays. Gene expression profiles in cells irradiated with 2 Gy were compared to those of non-irradiated cells using single-color microarrays. [Results] Twenty-two genes, including NFKBIA, responded in common to C-ions in all 6 cell lines, based on ANOVA filtering (P < 0.001). We found 173 genes that responded in common to C-ions in 4 cell lines. We identified many down-regulated genes (including the cell cycle–related genes CCNA2, CDCA8, CENPA, CRK7, ID1, KNTC2, TTK, and WEE1) that were more responsive to C-ions than X-rays among the 6 cell lines. In contrast, most of the up-regulated genes (including the p53 target genes ATF3, BTG2, CDKN1A, GADD45A, SESN1, TNFRSF6, and TP53INP1) showed similar responses to C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (P < 0.05). [Conclusion] Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle–related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays. Here, I would also discuss the dose-dependency of the induction of G2/M arrest and the expression cell cycle related genes.

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Methylations of DNA typically occur at CpG island in the promoter region of genes resulting in the decrease of transcription. DNA methylation status has been studied in association with carcinogenesis and aging. Biological relevance of changes of methylation pattern in the genomic DNA to cellular responses of chronic radiation exposure. In addition, whether the change of methylation pattern influences radiation sensitivity of cells or not is not determined. In order to understand the relationships between radiation exposure and the changes of methylation pattern, methylation sensitive arbitrarily primed PCR (MSAP-PCR) was performed using genomic DNA from parental HepG2 cells , other that from 3 sub-cell lines established by long-term exposure to X-rays, and that of radioresistant HepG2-8960-R cells. Compared with HepG2, we cloned 17 genomic regions where methyaltion status was changed in other sub-cell lines.. Of these, 4 regions were part of so-called genomic CpG island. Interestingly, 2 of them were related to the potassium ion channel (PIC). Since decrease of PIC activity is reported after exposure to low dose of X-rays, MSAP-PCR adopted in this study is suggested to be useful for the study of the relationship between DNA methylation and radiation exposure. We are now underway to investigate the changes of gene expressions cloned in this study and their biological significances.

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Previously we identified 65 radioresistant marker associated with heterogeneity in radioresistance among human tumor cell lines (The 49th annual meeting of the JRRS) (K. Ishikawa et.al., Int J Radiat Oncol Biol Phys. 2006). We have analyzed the expression of these markers in cervical cancers treated by radiotherapy.
Tumor samples were obtained from 25 patients with squamous cell carcinoma of the uterine cervix, before and during fractionated radiotherapy. Alterations in gene expression were analyzed by oligonucleotide microarray experiments (Codelink, GE Healthcare). When we applied the genes identified as radioresistant markers into clustering analysis of the patients, they were effective in separating the poor responders (local recurrence at 6 months or 1 year following radiotherapy) from good responders (complete remission at 6 months or 1 year). We found that in the poor responders there was a tendency for the genes expressed lower in the radioresistant cultured cells to be further suppressed, while those expressed at higher levels in the radiosensitive cultured cells were further induced in the good responders. Some genes harboring extra cellular matrix were included in this clustering.
Our data suggested that the radioresistant marker produced from in vitro would be useful for clinical analyses. This result gives some support to the application of in vitro analysis to in vivo analysis.

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Introduction: Cellular response to ionizing radiation is complex and the expression level of various genes related to DNA repair, cell cycle and cell death. Using boron-neutron capture method, we separately exposed Kupffer cells and endothelial cells in the mouse liver to α-particles and the change of gene expressions was analyzed by an oligonucleotide microarray. We found that irradiation induced a kind of inflammatory status in the liver. In order to confirm cell to cell interaction in the liver induced by radiation exposure, we examined the change of gene expressions in response to γ-radiation induced in primary culture cell which isolated Kupffer or hepatocyte cells in rat liver. Methods: Rat livers were digested by collagenase perfusion and parenchymal and non-parenchymal cells were separated using low speed centrifuge method. Adherent cells to the plastic dish were used as Kupffer cell fraction. Expression changes of cytokine genes were analyzed by real-time PCR 24 hrs after cells were exposed to 5 Gy of γ-rays. The genes whose expression levels were significantly different between irradiated and non-irradiated cells in our previous study using mouse livers were also analyzed. Result and discussion: Gene expressions of IL-1β and IL-6, pre-inflammatory cytokines, were dramatically up-regulated 3 hrs after radiation to Kupffer cells. Then expression level returned to basal level. Gene expression of anti-inflammatory cytokine IL-10 started increasing 3 hrs after radiation and reached the peak after 12 hrs. These results suggest that inflammatory status of the liver reached the peak 3 hrs after radiation and goes down to the sedation thereafter. Although Ak3 gene expression was down-regulated by all kinds of radiation treatments in the mouse microarray analysis, its expression was not changed in hepatocytes but was down-regulated in Kupffer cells after irradiation. Hpxn and Car3 gene expressions were up-regulated and down-regulated, respectively in the mouse microarray were not changed after hepatocytes were irradiatied. In Kupffer cells, both Hpxn and Car3 gene expressions were down-regulated 24 hrs after γ-ray radiation. These results suggested that biological influences of radiation induced in the liver are more attributed to Kupffer cells than parenchymal hepatocytes.

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The tumor suppressor p53 protects the cell against cancer by inducing apoptosis in response to multiple stresses. Some 50% of human cancers have mutations or deletions in the gene for p53 that inactivate or impair its apoptotic potential. On the other hand, in cancer therapy, p53 causes severe side effects by inducing excessive apoptotic death in several normal tissues. Thus, p53 inhibitory drugs have been suggested to rescue the acute response to cancer therapy. In this respect, recent studies have shown transcription-independent p53-mediated apoptosis via mitochondria. These findings indicate that effective p53 inhibitors should suppress both transcription-dependent and -independent pathways. We have reported a novel effect of sodium orthovanadate (vanadate) that inactivates p53 by inducing conformational change in it. Therefore, we investigated the effect of vanadate on the each pathways. We initially confirmed vanadate as an inhibitor of p53 transcription by luciferase reporter assay for its transcription and by immunoblotting of its transactivation. Furthermore, immunocoprecipitation analysis using anti-Bak, Bcl-2, and Bcl-xL antibodies demonstrated that vanadate suppressed the interaction of p53 with these Bcl-2 family proteins. Considering the evidence that these interacting proteins bind core domain of p53, vanadate may influence this domain, resulting in the inhibition of the binding of target genes and these protein partners.

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Although UV radiation has been known to induce apoptosis through an activation of signal transduction triggered by DNA damage, the responses of cells to UV radiation are quite different among different tissue sources. For example, UV causes a rapid cell death in most hematopoietic cell lines including human leukemic Jurkat cells and HeLa cells established from human cervical carcinoma show a delayed form of apoptosis following exposure to UV.
In order to know the mechanisms of cell-type dependent apoptosis, Jurkat and HeLa cells were irradiated with UV (254 nm), incubated for various time intervals after irradiation, and examined the changes in morphological and biochemical characteristics in the early stage. After UV irradiation, rapid appearance of small cells was observed in Jurkat cells, but not in HeLa cells. This phenomenon is a reflection of apoptotic volume decrease (AVR), which is observed in early stage of apoptosis and caused by the efflux of intracellular KCl. Interestingly, inhibition of K⁺ ion-channels with 2 mM 4-aminopyridine efficiently blocked the induction of apoptosis by UV radiation in Jurkat cells, but not in HeLa cells. These results suggest that UV irradiation causes K⁺ ion-channel alterations and triggers a rapid form of apoptosis in Jurkat cells.

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Mitogen-activated protein kinases (MAPKs) are activated through the kinase cascades of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). MAPKKK proteins relay upstream signals through the MAPK cascades to induce cellular responses. However, the molecular mechanisms by which given MAPKKKs are regulated remains largely unknown. Here, we found that a serine-threonine protein kinase (kinase X) physically interacted with the MAPKKK members MEKK1 and MEKK2 (MEKK1/2). The COOH-terminus, including the catalytic domain, but not the NH2-terminus of MEKK1/2 was necessary for the interaction with kinase X. Kinase X inhibited MEKK1/2 activation without preventing MEKK1/2 binding to its substrate, SEK1. Most importantly, kinase X suppressed the autophosphorylation of MEKK2. On the other hand, the knockdown of Kinase X by transfection with kinase X shRNA expression vector enhanced the stress-induced phosphorylation of the MAPKKK targets, MKK3/6. Taken together, our results indicate that kinase X negatively regulates the activation of MEKK1/2 by direct interaction with MEKK1/2's catalytic domain, suggesting a novel mechanism for MEKK1/2 regulation.

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ATM-p53 pathway plays a critical role in maintaining genomic stability through its participation in multiple cell cycle checkpoints. After irradiation, ATM protein kinase is activated and autophosphorylated at the sites of DNA double-strand breaks (DSBs). Phosphorylated ATM (pATM) forms the initial foci in the chromatin region flanking DSBs. Subsequently, the number of foci decreases concurrently with DSBs repair, while some fraction of foci remain persistently. Thus, it can be hypothesized that the persistent foci formation of DNA damage checkpoint factors is involved in ATM-p53-dependent permanent cell cycle arrest. In the present study, we established single-cell based assay to test the hypothesis. Normal human diploid cells were irradiated with ionizing radiation and plated onto cover slips at low density. After incubation for 3 days, pATM and 53BP1 foci were investigated by fluorescence microscopy. In this experimental setting, no micro-colony which all cells had foci was observed in rapidly growing colonies, but the frequency of cells with foci was sufficiently high in the fraction of non-dividing and slowly growing colonies. Foci size in non-dividing and slowly growing cells is larger than those of rapidly growing cells. These results suggest that large persistent foci of pATM and 53BP1 are sufficient for permanent cell cycle arrest of normal human diploid cells exposed to radiation.

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SMG1; PI-3 kinase and UPF1; RNA helicase are well known for their critical role in Nonsense Mediated mRNA Decay (NMD) pathway, which causes the elimination of mRNAs containing premature stop codons. However, their essential role in DNA metabolism (cell cycle progression and DNA damage sensoring) has been also demonstrated. Their function in DNA metabolism appears to be independent of NMD and SMG-1 plays important roles in the maintenance of both genome and transcriptome integrity in human cells. In this study, we analyzed the role of SMG1 on the expression p53 by RNAi. Accumulation of p53 by proteasome inhibitors reduced in SMG1 knock down cells and this seems to imply that SMG1 regulates the amount of p53.

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The movement of the nematode Caenorhabditis elegans in the presence of food (bacteria) decreases by up to approximately 60 % of that in environments without food (i.e., basal slowing response). Additionally, in preliminary experiments we demonstrated that γ irradiation also decreases the animals' movements.
In the present study, we aimed to verify and compare the effects of 2 distinct factors—the presence of food and γ irradiation—on the deceleration of movement in C. elegans. Adult animals (N2 strain) were irradiated with graded doses of γ-rays (100-1500 Gy) and their movement on agar plates with or without food was examined.
The movement of irradiated individuals on plates without food decreased in a dose-dependent manner and became approximately 40 % of that of non-irradiated one at 1500 Gy. On the other hand, no significant difference was observed between the movement of irradiated and non-irradiated individuals on plates with food up to doses of 1500 Gy. This result suggests that effects of 2 factors—presence of food and γ irradiation—on movement deceleration are not always additive. Furthermore, since, at each dose, the movements of the irradiated individuals on food were less frequent than those on plates with no food, it is suggested that the pathways transmitting information on the presence of food may be maintained to some extent following γ irradiation.

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Since the human breast carcinoma MCF-7 cells lack caspase-3 to play as an important factor in apoptosis, the loss of this protein is considerd to be involved in the radioresistance in MCF-7 cells. In this study, we have reported that the overexpression of caspase-3 enhance radiation-induced apoptosis. Furthermore, the involvements of other caspase and the apoptotic pathway through mitochondria and/or TNFα-family in this radiation-induced apoptosis were examined. The cDNAs of caspase-3 were inserted in the mammalian expression vector, pCI-neo. This plasmid was transfected into MCF-7 cells. After irradiation, the apoptotic cells were evaluated by morphological change by using PI staining. The overexpression of caspase-3 did not increase a number of apoptotic cells without irradiation. However, X-irradiation significantly induced apoptosis in the caspase-3-overexpressed cells in comparison with that in the mock cells. This result suggest that caspase-3 acts as an important role for the radioresistance of radiation-induced apoptosis in the MCF-7 cells. Moreover, to examine whether other caspases were involved in this apoptotic induction, inhibitory peptides against caspase-8 (Ac-IETD-CHO) and caspase-9 (Ac-LETD-CHO) were utilized. Not only Ac-LETD-CHO but also Ac-IETD-CHO suppressed the enhancement of radiation-induced apoptosis in the caspase-3-overexpressed cells. This fact suggested that the radiation-induced apoptosis was associated with both apoptotic signal transduction pathways through mitochondria and TNFα-family.

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The CD180, a cell surface receptor of lipopolysaccharide (LPS) and a homologue of Toll-like 4(TLR4) on B lymphocyte, has been revealed as suppressing molecule of radiation-induced apoptosis in vitro. However, few studies regarding the functional mechanism of the CD180 molecule associated with radiation-induced apoptosis have been reported in vivo. In the present study, we have examined the effect of irradiation on the expression patterns of CD180 and apoptosis of spleen cells associated with the expression of CD19 and CD22 in vivo. BALB/c female mice (7-8 weeks) were irradiated with X-rays at 150 kVp and 20 mA at a dose rate of 5.4 Gy/m. Spleens were removed after whole body 4 Gy-irradiation. Histochemical analysis demonstrated that the number of CD180 positive- B lymphocytes was significantly increased at 6 h after irradiation when compared with both 3 h after irradiation and 0 h (sham irradiation), irrespective of no significant change of the numbers of B lymphocytes positive for CD19 or CD22 after irradiation for 0 h, 3 h, and 6 h. To clarify the relationship of the expression patterns between apoptosis and CD180, double staining for TUNEL and CD180 was performed and results showed that the number of CD180-negative cells undergoing apoptosis was less than that of CD180-positive cell undergoing apoptosis at 6 h after irradiation. These results suggested that CD180-positive B lymphocytes were resistant to apoptosis induced by irradiation in vivo.

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Objective: To examine the predictive value of FGF2 in expression for disease failure after chemoradiotherapy in cervical cancers. Patients and methods: The subjects were 28 patients with cervical cancers. A punch biopsy before treatment (pre) and a second biopsy from the same site of the cervix (mid) were taken one week after the start of therapy, when patients had received 9 Gy of whole pelvic irradiation and a single dose of CDDP, and branchytherapy had not begun. In addition to FGF2, as recent reports proposed a close relationship among FGF2, laminin, and CD44, expression of CD44, and laminin was also analyzed using a streptavidin-biotin immunoperoaidase technique. Positive area proportion (%) of FGF2 and CD44 were analyzed using an image analysis system, and laminin staining pattern was scored by continuity of the basement membrane immunopositivity. Patients were defined as good (n =18) or poor responders (n =10) based on their two-year disease-free survival. Result: Protein expression of FGF2 in midtreatment samples (mid) was significantly higher than in pretreatment samples (pre) (P = 0.030). The ratio change (mid versus pre) of FGF2 expression in poor responders was significantly lower than that in good responders (P = 0.037). The number of cases with discontinuity of laminin staining pattern at pre was significantly increased in the poor responders (P = 0.046). Protein expression of CD44 was not significantly changed between good responders and poor responders. An association was found between laminin staining pattern in pre and ratio change of FGF2 or CD44 expression in mid (P = 0.026 and P = 0.036, respectively). Conclusion: We found that FGF2 ratio change and laminin staining pattern at pre were significantly associated with prognosis. These molecular features might help us to identify patients at high risk of disease failure after CRT.

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Purpose: As a foundation for improving cancer radiation treatment, we have been trying to find novel proteins involved in radio-resistance. In this study, we performed proteomic analysis to search for nuclear proteins of which expression levels differ pre- and post-X-ray irradiation. We focused on clarification of the involvement of aldolase A, one of the candidate proteins identified by the analysis, in radio-resistance. Methods: Fluorescent two-dimensional differential gel electrophoresis (2-D DIGE) was performed to identify proteins that show concentration changes after exposure to X-rays (5Gy) in a nuclear fraction of HeLa cells. Expression of aldolase A in HeLa and UV^r-1 cells was suppressed using its specific siRNAs, and the sensitivity of the suppressed cells to X-ray-induced cell death was analyzed by colony survival assay. Results and discussion: Twenty-four hours after X-ray irradiation of HeLa cells, expression levels of 6 proteins were increased in the nuclear fraction; these proteins included aldolase A, a glycolytic enzyme commonly localized in cytosol. When expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray induced cell death was observed. In addition, expression of aldolase A was higher in a derivative radio-resistant cell line, UV^r-1 , than in its parental cell line, RSa. These findings suggest that high levels of aldolase A confer radio-resistance and that aldolase A may play other roles in addition to its role in glycolysis. It is also suggested that suppression of aldolase A expression before irradiation may improve the efficiency of anti-cancer radiation therapy.

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