Mitsuru Nenoi

Gene Expression Profiles in Mouse Liver after Long-Term Low-Dose-Rate Irradiation with Gamma Rays

Abstract Changes in gene expression profiles in mouse liver induced by long-term low-dose-rate &ggr; irradiation were examined by microarray analysis. Three groups of male C57BL/6J mice were exposed to whole-body radiation at dose rates of 17–20 mGy/day, 0.86–1.0 mGy/day or 0.042–0.050 mGy/day for 401–485 days with cumulative doses of approximately 8 Gy, 0.4 Gy or 0.02 Gy, respectively. The gene expression levels in the livers of six animals from each exposure group were compared individually with that of pooled sham-irradiated animals. Some genes revealed a large variation in expression levels among individuals within each group, and the number of genes showing common changes in individuals from each group was limited: 20 and 11 genes showed more than 1.5-fold modulation with 17–20 mGy/day and 0.86–1.0 mGy/day, respectively. Three genes showed more than 1.5-fold modulation even at the lowest dose-rate of 0.04–0.05 mGy/day. Most of these genes were down-regulated. RT-PCR analysis confirmed the expression profiles of the majority of these genes. The results indicate that a few genes are modulated in response to very low-dose-rate irradiation. The functional analysis suggests that these genes may influence many processes, including obesity and tumorigenesis.

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays.

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

Estimation of interaction function gamma(x) with sparsely ionizing radiation.

The interaction function gamma(chi), which was introduced in the theory of dual radiation action as the probability that two energy transfers separated by distance chi combine with each other to produce a lesion, was estimated with sparsely ionizing radiation (60Co gamma rays and 40 kV X rays). Gamma(chi) was deduced on the assumption that the sensitive matrix is made up of small spherical flocculi distributed over the cell nucleus. The diameter of a flocculus was estimated at (4.0-11.2) X 10(-8) m when the diameter of the cell nucleus d was assumed to be 5 microns, and (4.0-11.4) X 10(-8) m when d was assumed to be 10 microns. It seems reasonable to hypothesize that the flocculus corresponds to the linker DNA in the chromatin structure of DNA, because the size of the linker DNA as a target (about 40 nm) is consistent with the diameter of flocculi obtained in this study.

Adaptive response in embryogenesis: VI. Comparative microarray analysis of gene expressions in mouse fetuses

Purpose: Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. This phenomenon, called radiation-induced adaptive response (AR), has been described in a wide range of biological models. We previously demonstrated the existence of AR in mice during late organogenesis. In this study, we investigated molecular mechanisms underlying AR in this model. Materials and methods: Using DNA microarrays, we performed a global analysis of transcriptome regulations in adapted and non-adapted cells collected from whole mouse fetuses, after in utero exposure to priming irradiation. Results: We identified AR-specific gene modulations. Our results suggested the involvement of signal transduction and Tumor protein (p53)-related pathways in the induction of AR. Conclusions: Our results are in agreement with previous investigations showing that AR could be dependant on p53 activity. The observed gene modulations may also have possible consequences for subsequent developmental process of the fetus. This is the first report of AR-specific modulations at the molecular level in utero, which could serve as a basis for subsequent studies aimed at understanding AR in this model and possible long-term effects.

Involvement of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of ionizing radiation

CDKN1A is a cyclin-dependent kinase inhibitor that plays a critical role in cell cycle checkpoint regulation. It is transcriptionally induced by TP53 (p53) following exposure to ionizing radiation (IR). Induction of CDKN1A after irradiation is closely related to IR-sensitivity of tumor cells, but the underlying mechanisms remain obscure because conventional reporter gene systems respond poorly to IR unless hyperlethal doses are used. Here, we performed a promoter analysis of the CDKN1A gene following irradiation with clinically relevant doses of IR using the adeno-associated virus-mediated reporter system which we have recently shown to be highly responsive to IR. We demonstrate that there are regulatory elements at -1.1 kb, -1.4 kb, and -1.8 kb, and deletion of these elements attenuate induction of the CDKN1A gene promoter in response to 0.2-2.0 Gy of IR. EMSA and ChIP assays showed that Oct-1 binds constitutively to the elements at -1.1 kb and -1.8 kb. Functional involvement of Oct-1 was confirmed by RNA interference targeting the Oct-1 gene, which suppressed both the basal and IR-inducible components of the CDKN1A expression. Thus, our results reveal that Oct-1 is crucial to the TP53-mediated regulation of the CDKN1A gene promoter following exposure to clinically relevant doses of IR.

Interaction Function γ(x) for Chinese Hamster Cells Treated with Hypertonic Phosphate-Buffered Saline after Irradiation

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.

Modulation of Gene Expression After Exposure to Ionizing Radiation

Regulation of gene expression is one of the fundamental mechanisms by which cells utilize the information in their DNA to obtain diverse characteristics, such as ability of differentiated cells to play specific roles and ability to respond to extracellular stresses including ionizing radiation (IR). The research on transcriptional regulation of genes after exposure to IR has a long history tracing back to early studies in late 1980’s. In those studies, expression of individual genes was separately measured by a classical hybridization method. Recently, however, functional genomics approaches, such as microarray profiling, enable us to simultaneously monitor the expression of thousands of genes, and are now recognized as a firmly established methodology in radiation biosciences. The porpose of investigation of transcriptional gene regulation by IR is primarily to gain an insight into how the human bodies respond to IR and eventually how radiation hazards develop. IR-induced hematopoietic death is an example where proapoptotic gene expression is enhanced in radiosensitive hematopoietic stem cells after exposure to high doses of IR, which causes untolerable loss of peripheral blood cells, ultimately resulting in the individual death. Secondary, the purpose of the reserach on transcriptional gene regulation is to search for biomarkers which indicate the quantitative IR-exposure records. Such biomarkers may help us estimate the exposed dose of public peolpe in the emergency cases. For these purposes, studies have been extensively carried out by lots of researchers, and plenty of insights were obtained. In this review article, some of these studies, including those carried out in our laboratory, will be overviewed with an emphasis on the important roles of a tumor supressor p53 in transcriptional regulation after IR exposure and organ-dependence of transcriptional regulation in vivo. Methodology for the research on transcriptional regulation is also briefly touched. And the future perspectives of the research on transcriptional responses to IR will be discussed.