Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: TGF-ß Protects Human Mammary Epithelial Cells from Radiation-Induced Centrosome Amplification

Authors: Mary Helen Barcellos-Hoff, Bahram Parvin, Anna C. Erickson and Rishi Gupta

Institutions: Department of Cell and Molecular Biology, Life Sciences Division, Ernest Orlando Lawrence, Berkeley National Laboratory, Berkeley, California 94720

In recent studies we have shown that ionizing radiation (IR), a known carcinogen of human and murine mammary gland, compromises human mammary epithelial cell (HMEC) polarity and multicellular organization in a manner characteristic of neoplastic progression through a heritable, non-mutational mechanism (1). Thus, when all cells are irradiated with a significant dose (2 Gy), the daughters of irradiated cells lose their ability to interact with each other and the microenvironment.
We have postulated that abnormal cells may accumulate under these circumstances and would then contribute to the development of neoplasia in vivo (2). To test this we determined the frequency of centrosome defects, which frequently accompany tumor progression, in HMEC as a function of radiation dose (0.1-5 Gy). Non-malignant S1 HMT-3522 HMEC were seeded as monolayers and subjected to IR 4 hours post plating. Daughters of the surviving cells were analyzed for centrosome
abnormalities six days later by immunofluorescent staining for (-tubulin. IR increased the frequency of S1 cells with 3 or more centrosomes as a function of radiation dose up to 2 Gy.

The irradiated HMEC phenotype is augmented by TGF-ß which is rapidly activated in response to IR in mouse mammary gland (3) and plays a critical role in epithelial cell fate decisions (4). TGF-ß can either suppress or promote tumor progression via a variety of mechanisms (5). We asked whether TGF-ßaffected radiation-induced centrosome instability. Culture with additional TGF-ß (400 pg/ml) following radiation exposure decreased the frequency of surviving HMEC with abnormal centrosomes numbers. Consistent with our studies in irradiated mice, irradiated HMEC also activate more TGF-ß than control cells. Addition of TGF-ß neutralizing antibodies to irradiated cells resulted in increased frequency of cells with centrosome amplification. Thus, TGF-ß plays a dual role in response to IR by protecting against genomic instability that would be generated by radiation-induced centrosome amplification, while promoting phenotypic neoplastic progression.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Secretory clusterin (sCLU) expression as a sign of genomic instability, and a potential promoter of instability in bystander cells.

Authors: T. Criswell, M. Beman, S. Araki, K. Leskov, D. Klokov, and DA.
Boothman.

Institutions: Departments of Radiation Oncology, Pharmacology & Pathology, Lab of Molecular Stress Responses, Case Western Reserve University, Cleveland, OHIO 44106-4942; dab30@po.cwru.edu

Expression of the secretory form of clusterin (sCLU) is a sensitive marker of genetic instability created after extremely low doses of ionizing radiation (IR). In fact, up-regulation (>2-fold) of sCLU mRNA and protein in response to IR doses as low as 2 cGy have been reported (Yang et al., PNAS, 2000; Criswell et al., Cancer Biology and Therapy, 2003), with >200-fold after 1 Gy or more, 48-72 h after exposure. The CLU promoter is so responsive to low doses of IR that we are exploiting IR-inducible sCLU levels as a sensitive measure of damage, and have developed a ‘biodosimeter’, wherein real-time bioimaging of CLU promoter activity using human MCF-7 breast cancer cells with a stably integrated CLU promoterluciferase reporter construct in culture or as xenografts in nude mice can be monitored.

A transgenic animal with an incorporated CLU promoter-luciferase cassette that acts as a sentinel of low dose IR or other agent exposure, is also being generated. Unlike the pro-death nuclear form of the clusterin protein (nCLU) (Yang et al., NAR, 1999; PNAS, 2000; Leskov et al., 2001; JBC, 2003), the secreted form of clusterin (sCLU) is a pro-survival factor, as recent small interfering RNA (siRNA) gene repression studies from our laboratory have demonstrated (Fig. 1, Leskov et al., In Prep., 2004). Since large amounts of sCLU are secreted into the media and sera of cells and tissues, respectively, after IR (Klokov et al., In Press, 2003), the downstream bystander effects of sCLU could further contribute to a cascade of IR-induced effects, including in non-irradiated cells. Thus, improved understanding of the regulation and functions of sCLU is important for explaining the longterm genetic instability effects of IR in human cells after low doses of IR. This is further highlighted by the inverse relationship between functional p53 and sCLU levels, wherein loss of functional p53 results in dramatic elevations of basal and IR-inducible sCLU levels that we believe further contributes to the survival of IR-treated, genetically unstable cells (Criswell et al., Cancer Biology and Therapy, 2003).

Recent data from our laboratory indicate a complex regulatory pathway of sCLU gene expresson in response to low doses of IR. We are also investigating the downstream effects of sCLU secretion into the media or sera, and we hypothesize that this protein is a major factor in bystander effects reported by others in response to IR, or other types of cytotoxic stress. Our current data indicate the following regulatory processes are stimulated in human cancer and normal cells after low doses of IR that control sCLU gene expression.

sCLU signal transduction processes: Treatment of human MCF-7 or HCT116 p53-/- cancer cells or p53 mutant CT-5 mouse embryonic fibroblast (MEFs) cells with IR doses of 0.02-10 Gy results in the activation of the c-src and p38 MAPK signal transduction pathways, and expression of sCLU. Co-treatment of cells with chemical inhibitors of these pathways prevented sCLU endogenous expression, as well as inhibited CLU promoter-luciferase activities. Forced transient expression of kinase dead c-src or P38 MAPK also suppressed sCLU expression 48-72 h post-IR treatment. Thus, IR stimulated c-src and p38 MAPK appear to regulate downstream sCLU expression. Experiments are underway to elucidate these signal transduction pathways, as well as the functional significance of abrogating these pathways in terms of cell survival and genetic instability (Criswell et al., In Prep., 2003).

The signal transduction process above can be suppressed by wild-type p53. A direct comparison between HCT116 parental cells expressing wild-type p53 and isogenic p53-/- HCT116 cells revealed that only cells lacking functional p53 induced sCLU. Expression of E6 (that binds and degrades p53) resulted in higher basal sCLU levels, and a more pronounced IR-inducible level of this secreted protein. IR-inducible expression of sCLU and regulation of this protein by p53 was not a result of altered cell cycle checkpoint regulation, since IR-treated HCT116 parental and p21-/- HCT116 cells did not differ in their expression of sCLU (Criswell et al., CBT, 2003).

We are currently testing the theory that IR-inducible levels of sCLU can abrogate TGF-ß1 cell signaling. We recently showed that exposure of wild-type TGF-ß1 RII receptor-containing human colon or breast cancer cells with TGF-ß1 caused dramatic expression of sCLU, with time-course responses identical to those following IR, and growth suppression. In contrast, genetically matched cells lacking the RII receptor were non-responsive to TGF-ß1 treatments in terms of growth suppression and up-regulation of sCLU. Thus, TGF-ß1 can regulate sCLU expression in these cells. Interestingly, the presence or absence of the TGF-ß RII receptor did not affect sCLU protein level induction(Sang’s data?). This indicates that sCLU expression can be regulated by TGF-ß1 or IR, but the regulatory signal transduction processes are different. Since sCLU can bind the RI and RII receptors of TGF-ß1, we hypothesize (and are currently testing the theory) that sCLU represents a negative feedback loop acting to suppress normal TGF-ß1-mediated growth and gene regulation in irradiated or non-irradiated ‘bystander’ cells. This work was supported by DOE grant DE-FG-022179 to DAB.

References:
Criswell, T, Klokov, KS, and Boothman, DA.(2003) Transcriptional repression of clusterin by the p53 tumor suppressor protein. Cancer Biology and Therapy, 2(4): 25-31.

Criswell, T., Leskov, K., Miyamoto, S., Luo, G-B., and Boothman, DA. (2003) IR-inducible transcription factors in mammalian cells at clinically relevant doses. Oncogene, 22(37): 5813-5827

Klokov D, Criswell T, Sampath L, Leskov K, Frinkley K, Araki S, Beman M, Wilson D, and Boothman, DA. Clusterin: a protein with multiple functions as a potential ionizing radiation exposure marker. In: 1st Nagasaki Symposium of
International Consortium for Medical Care of Hibakusha and radiation Life sciences. (Shibata Y, Yamashita S, Watanabe M, Tomonaga M Eds.) Elsevier, Amsterdam, In Press, 2003.

Klokov, D, Kang, S-W, Criswell, T, and Boothman, DA.(2003) Regulation of secretory clusterin levels by TGF -ß1. Cancer Research, In Prep.,

Klokov D, Sampath L, Frinkley K, Wilson D, and Boothman, DA. (2003) Development of a sensitive biodosimeter for the detection of low doses of ionizing radiation. In Prep.
.
Leskov, K, Antonio, S, Criswell, T, Yang, C-R, Kinsella, TJ, Boothman, DA.(2001) Rad. Research 156: 441-442

Leskov, K., Criswell, T.A., Antonio, S. Li, J., Yang, C-R., Kinsella, T.J., and Boothman, D.A. (2001) When X-ray-inducible proteins meet DNA double strand break repair. Seminars in Radiation Oncology 11: 352-372

Leskov, KS, Klokov, DY, Li, J, Kinsella T J, and Boothman, DA. (2003) Synthesis and functional analyses of nuclear clusterin: a cell death protein. J. Biol. Chem. 278: 11590-11600


Sun, W, Sawada, M, Hayes, P, Leskov, K, Boothman, DA, and Matsuyama, S. (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nature Cell Biology 5: 320-329

Yang, C-R, Odegaard, E, Leskov, K, Hosley-Eberlein, K, Criswell, T, Kinsella, TJ, and Boothman, DA. (200) PNAS, USA, 97: 5907-5912

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: 12.5 keV Xray Microbeam Bystander Studies With Human Mammary Epithelial Cells and Fibroblasts

Authors: E. A. Blakely¹, R. I. Schwarz¹, A. C. Thompson², K. A. Bjornstad¹, P. Y. Chang^1,3 C.J. Rosen1, and D. Sudar¹

Institutions: Divisions of ¹Life Sciences and ²Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720-8174 USA and ³SRI International, Menlo Park, CA 94025 USA.

We are using a novel X-ray Microprobe Beamline at the Advanced Light Source (ALS) at LBNL to investigate bystander effects of low doses in wellcharacterized human mammary epithelial cells (HMEC) and human skin fibroblasts (HSF). The ALS facility is capable of producing a beam of 12.5 keV X-rays with a focussed spot size of __m_ and a wide range of doses and dose-rates. Unlike normal X-ray sources, this beam has a very small background of either low- or high-energy X-rays. In initial studies, cultures grown in microwell slide chambers have been irradiated with precise stripes of dose up to 100_m wide. We are using fluorescence microscopy on a high-precision-controlled microscope stage to evaluate several classes of radiation-induced signals, how these signals are communicated across cell compartments, and how radiation changes cell signaling both acutely and chronically. We are investigating the radiation induction of p21Cip1 (CDKN1a), and phosphorylation of H2AX and p53 serine-15 as endpoints. Our preliminary results indicate that there is a dose- and celltype- dependent expression of p53 serine-15P within 10 minutes after exposure to a ____m wide stripe of dose. Immunohistochemistry of p53-serine-15P-positive cells traversed by the beam illuminates the path of the X-ray microbeam, with epithelial cells responding more rapidly and with greater intensity than fibroblasts. The intensity of the immunofluorescence scales with the dose. Using stripes of dose we are able to evaluate the spatial dependence of intercellular bystander effects. The number and fluorescence intensity of p53- serine-15P-positive cells in the unirradiated cell populations at perpendicular distances away from the dose stripe are being scored as a measure of the bystander effect, and compared to appropriate controls. We will report on cellular responses to doses of 400 cGy down to 10 cGy examined in a time course from 10 min to 6 hours after exposure.

This work was supported by the U. S. DOE's Low Dose Radiation Research Program under Contract No. DE-AC03-76SF00098.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Mechanistic Modeling of Bystander Effects: An Integrated Theoretical & Experimental Approach -Effects in PKcs suppressed AG 1522 cells-

Authors: L. A. Braby, D. Perez, E. A. Repnikova, and J. R. Ford

Institutions: Texas A&M University College Station TX 77843

The primary objective of this project is to provide data for building a mathematical model of the role of repair in the production of DNA damage in directly irradiated and bystander cells. Major tools in this study are the micronucleus assay and use of cells with different levels of expression of DNA-PK, resulting in different repair capacity. Current efforts are focused on using post-transcriptional gene silencing techniques to produce test populations of AG 1522 cells which express different levels of DNA-PK catalytic subunit. Using established protocols developed for other cell types, we found that DNA-PK could be reduced, but not completely eliminated in all cells of a culture. Thirty percent of the cells exhibited no DNA-PK expression by immunohistochemistry while the remaining 70% exhibited DNA-PK reductions of from 10-50% of the controls. While continuing to conduct experiments using the current optimized procedure, we are also continuing to search for more efficient ways to suppress DNA-PK.

Using the current small interfering RNA technique we are pursuing two specific research areas, micronucleus rates in bystanders of cells irradiated by 60 kV electrons, and evaluating the PK status of micronuclei induced in populations containing normal and reduced PK cells. Since we have a population of cells with various levels of PK activity, it should be possible to determine if the PK deficient cells show a higher probability of producing stable DNA damage at a given dose (as indicated by formation of a micronucleus) than do PK normal cells. This can be checked by use of an antibody for PK and analyzing (by fluorescent microscopy) nuclei and micronuclei for PK. Because of the spatial density of the cells in these experiments it is not possible to make a positive correlation between a micronucleus and the two daughter nuclei that produced it. However, it is possible to sample areas of the cell population (each containing several nuclei and one or more micronuclei) and make a statistical correlation between the fraction of nuclei expressing DNA-PK and the number of micronuclei produced. A correlation between number of PK negative nuclei and the probability that the micronuclei will be DNA-PK negative can also be explored. Assuming that micronuclei produced by PK positive nuclei would also be PK positive, and that we can reliably distinguish positive and negative micronuclei, the ratios of positive to negative nuclei and micronuclei should indicate the correlation between PK status and micronucleus induction. Preliminary results with this newly developing technique indicate that the number of PK positive and PK negative micronuclei per cell is about equal in PK suppressed populations. However, the number of parent cells that were PK positive significantly exceeded the numbers that were PK negative. If this relationship holds when the data are analyzed on a cluster by cluster basis, it would suggest that PK negative cells experience a higher rate of DNA damage leading to deletions that are large enough to be detected as micronuclei than do PK normal cells.

Previous experiments using antibody assays for DNA repair proteins in bystander cells on dishes which had a small fraction of the cells irradiated by 60 kV electrons were negative. No significant protein expression bystander effects could be detected, even though relatively high doses (up to several gray) were delivered to the cells in an irradiated area of approximately 0.03 by 1.0 cm. This contrasts with results obtained at the Gray Laboratory where significant frequencies of micronuclei were detected in bystander cells when one or a few cells were irradiated with similar doses of very low energy x-rays. Although the energy deposition patterns of carbon K X rays and 60 kV electrons are quite different, it is also possible that the difference in the endpoint was responsible for the difference in the results. To further explore this possibility, two 0.03 cm wide strips were irradiated across 1.5 cm diameter clusters of AG 1522 cells. Doses of approximately 1 and 5 Gy were delivered in the irradiated strips. Micronucleus frequency is being measured inside the irradiated strips and as a function of distance from the irradiated strip. Although the results are not yet complete, there is a strong indication of bystander cells expressing micronuclei. For this experiment, micronuclei were assayed 72 hours after irradiation. The preliminary results show that un-irradiated (bystander) cells on partially irradiated dishes produce about twice as many micronuclei per cell as did the cells on sham irradiated dishes (0.9% versus 0.5%). The rate of micronucleus production in bystander cells is approximately ten-percent of the rate in directly irradiated cells, but because of the relatively large number of bystander cells, there are nearly the same numbers of micronuclei produced in irradiated and bystander cells, after spontaneous
rates have been subtracted.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Apoptosis in Unirradiated Cells in Human Tissue as a Response to Radiation Damage in Cells up to 1 mm Away

Authors: Oleg V. Belyakov, Gerhard Randers-Pehrson, Stephen A. Mitchell, Stephen A. Marino & David J. Brenner

Institutions: Center for Radiological Research, Columbia University, New York, New York 10032

A central tenet of mutagenic and carcinogenic investigations has been that the relevant endpoint results from direct damage to the DNA of the initially affected

cells. A range of evidence has now emerged that suggests the importance of the other pathways, in particular, the so-called "bystander effect" where responses are observed in cells which are not directly traversed by ionizing radiation, but are located in the vicinity of directly irradiated cells.

Using the Columbia microbeam, we demonstrate for the first time a long-ranged bystander response in intact 3-D normal human skin tissue. The skin tissue systems are EpiDerm-200, modeling the keratinocyte containing epidermis, and EpiDerm-FT, a "full thickness" skin model with a fibroblast-containing dermal layer and a keratinocyte-containing epidermal layer.

The bystander effect we see is about a 3-fold increase in apoptotic rates in unirradiated epidermal keratinocytes located up to a distance of approximately 1 mm from irradiated cells in the same tissue.

The effects appear cell-type dependent: for example, signals from dermal fibroblasts do not appear to produce a bystander response in adjacent epidermal keratinocytes. It does seem clear, however, that bystander responses need to be taken into account when extrapolating radiation risks to very low doses when only a small fraction of cells are directly hit: simple extrapolation based on the number of cells directly hit may well be inadequate.

 

Supported by the Low-Dose Radiation Research Program of the U.S. Department of Energy.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Low Dose Radiation Research: Outreach and Resources

Authors: Antone L. Brooks and Lezlie A. Couch

Institution: Washington State University Tri-Cities, Richland, Washington 99338

The primary goal of this project is to develop and maintain a scientifically valid Website that provides information regarding the status of current research on biological responses to low doses of ionizing radiation. One aim of the Website is to provide scientific information that is easy to understand and can be used by the educated public on issues related to the biological changes and health effects induced by exposures to low doses of ionizing radiation. This is done through direct interactions with the public, producing an up-to-date series of slide shows on radiation, defining terms associated with radiation, linking to other web sites of similar interest and providing other features that are useful for helping the interested public to understand low dose radiation. Another aim of the Website is to provide a useful communication link between scientists funded by the DOE Low Dose Radiation Research Program. The Website includes a complete listing of the past and currently funded Program projects, the publications that resulted from the research conduced in the program, links to other agencies that fund research in similar areas and a listing of current and future meetings related to radiation research. The second goal of the project is to use additional forms of communication to help facilitate the dissemination of the scientific information generated from the radiation research. This is done through presentations to the public, presentations and participation in scientific meetings both inside and outside of the field of radiation biology, facilitating the organization of sessions about low dose radiation effects in scientific societies, and publications in the scientific literature on exposure and responses to low doses of radiation. Dr. Brooks is a member of a number of scientific organizations, has been appointed to scientific councils, serves on editorial boards and is a peer reviewer for scientific journals in the field of radiation biology. He also provides scientific input into public and scientific committees that are concerned with the regulation of radiation exposure and standards. The Website maintained by this project is a Washington State Website and can be found at http://www.lowdose.energy.gov. Comments on the Website, suggestions for Website improvements and outreach opportunities to other scientific societies or organizations can be addressed to Dr. Brooks at tbrooks@tricity.wsu.edu, phone 509-372-7550 or fax 509-372-7552.

This research was supported by the Office of Biological and Environmental Research, U.S. Department of Energy through a grant No. DE-FG03-99ER62787 to Washington State University.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Gene Expression Profile of Normal Human Fibroblast After Ionizing Irradation, a comparison study between low dose and high dose.

Author: D.Chen

We have carried out study to investigate global gene expression changes of G1/G0 arrested normal human fibroblast subject to ionizing radiation. Using cDNA microarray made with 7458 named human cDNA clones, we focused on differential gene expression for a low-dose X-ray irradiation at 2cGy and its comparison with high-dose at 4Gy. Four time points were studied at 1hr, 2hr, 4hr and 24hr after irradiation. Three independent experiments were performed for each dose/time point. After significant analysis, we found that a remarkable number of genes are changed after irradiation at both low and high doses. The percentage of changed genes at 1hr, 2hr, 4hr and 24hr after radiation are 0.48%, 5.61%, 1.35% and 1.77% at 2cGy; and 1.9%, 31.7%, 2.69% and 8.7% at 4Gy respectively. When comparing differences between low and high doses, we found that, although some of the genes are changed at different time point between the 2 doses, 251 genes appears to be differentially expressed in both doses. There are 174 genes that are uniquely changed at only low dose and 1907 genes are only changed at high dose at all the time points. The most dominating functional groups that are changed after irradiation are more diversely distributed in low dose, i.e., twice of the number as they are in high dose. Among these functional groups, in the low dose there is little currently known stress responding categories in low dose, whereas in high dose, we can find several stress-related groups like DNA packaging, mitotic checkpoint, cell growth and maintenance, chromosome organization and biogenesis. Principal component analysis reveals that there is a dominant expression pattern against the time course. The magnitude of changes is at peak at 4hr in low dose whereas it is at 2hr in high dose. This is indicates a delayed response for low dose in comparison with high dose irradiation.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Genome-scale modeling of low-dose radiation responses using microarray-based gene networks.

Authors: MA Coleman¹, T Cricthlow²¹, D Nelson¹, L Peterson³ and AJ. Wyrobek¹,

Institutions: ¹Biology and Biotechnology Research Program. ² Center for Applied Scientific Computing, Lawrence Livermore National Laboratory, Livermore, CA. 94551. ³Departments of Medicine, Molecular and Human Genetics, and Urology, Baylor College of Medicine, Houston, TX 77030.

The identification and characterization of regulatory elements of ionizing radiation (IR)- responsive genes can provide valuable understanding of the genetic mechanisms of IRresponse. Groups of genes with apparently different functions have been shown to have similar IR response patterns. Similar radiation response phenotypes are predicted to have common IR-induced gene expression profiles that are controlled by shared groups of regulatory elements. Using gene expression microarray data in conjunction with tools developed for DNA sequence/pattern recognition we have built a gene-network model that groups promoters and identifies their regulatory elements that control differential aspects of cellular responses to IR. Our model used differential IR radiation responses to varying doses between 1 cGy and 400 cGy to identify known effector genes of the TP53 damage sensing/signaling pathway that share the following common transcription factor binding sites (TAFs): EGR, ETS, MAZ, MZ1, SP1, and ZBP. Combinations of TAFs can be grouped into modules that define the IR-responsive promoter. In the case of the TP53- damage sensing pathway, different combination of the regulatory elements EGRF-ETSMAZF- ZBPF and their relative locations to each other were found to be conserved among modulated genes such as: GADD45A, CDKN1A, PCNA. The shared promoter elements identified in silico is conserved across species such as human and mouse, suggesting common mechanisms of IR-responses. This model is now applicable to identify novel IR-modulated genes based solely on TAF homology searching. Data to identify genes/pathways that are associated with different radiation response phenotypes (e.g., low dose sensitivity, adaptive response, sensitivity to chromosome damage, etc.) will require larger data sets that account for dose, time, tissue specificity and genetic variation. The identification and characterization of regulatory elements of IR-responsive genes will provide powerful biological indicators of genetic susceptibilities for tissue and genetic damage.

This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48 with funding from the DOE Low Dose Radiation Research Program.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title:Microarray transcription profiles for adaptive response in human lymphoblastoid cells identify molecular linkages between DNA damage and signal transduction

Authors: MA Coleman¹, F Marchetti1, D Nelson¹, LE Peterson², E Yin¹, and AJ Wyrobek¹.

Institutions: ¹Biology and Biotechnology Research Program. Lawrence Livermore National Laboratory, Livermore, CA. 94551; ²Departments of Medicine, Molecular and Human Genetics, and Urology, Baylor College of Medicine, Houston, TX 77030.

Previous studies have shown that a low dose of ionizing radiation (IR) can induce protection from a subsequent high dose of IR, but the responsible genes and pathways are not well understood. We applied gene transcript profiling in combination with micronucleus assays to elucidate the molecular pathways underlying the cytogenetic radioadaptive response in human lymphoblastoid (HLB) cell lines. HLB cells received a priming dose of 5 cGy followed 6 hr later by a challenging dose of 200 cGy or the challenging dose only. Two HLB cell lines that demonstrated reproducible radioadaptation by the micronucleus assay were compared to a HLB cell line that reproducibly did not adapt using Affymetrix U95A oligonucleotide microarray chips representing ~22,000 genes. RNAs were isolated 4 hours after the challenging doses in experiments that simultaneously tested for adaptation and non-adaptation. Statistical analysis with a false discovery rate of <0.1 and a significance value of >0.05 identified 166 genes among the three cell lines with significant differences in transcription levels after exposure to fractionated dose (5 and 200 cGy) versus 200 cGy only. Also, cluster analysis identified consistent differential responses between the two adapting cell lines and the non-adapting cell line. Genes associated with adaptive response by cluster analysis include ATM, JUND, c-MYC as well as SP100, M phase phosphoprotein, INF2AR and a member of the HSP70 family. A global view of radioadaption using Gene Ontology Maps suggests that radioadaptation may be linked to modulated expression of genes associated with signal transduction, inflammation, and stress response. Interestingly, genes involved in protein synthesis were found to be highly induced within all three HLB cell lines in response to the low-dose followed by the high dose. These data suggest a relationship between pathways involved in signal transduction and DNA damage sensing and repair. Further studies are needed to investigate the time course of gene expression profiles after the priming dose but before the challenging dose to identify and gain insights into the cellular functions and pathways of genes that may predispose a cell to radioadaptation.

This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48 with funding from the DOE Low Dose Radiation Research Program.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Use of Computational Modeling to Evaluate Hypotheses about the Molecular and Cellular Mechanisms of Bystander Effects

Authors: Yuchao “Maggie” Zhao and Rory Conolly

Institutions: CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park North Carolina 27709, USA

A detailed understanding of the biological mechanisms of radiation-induced damage at the molecular and cellular levels is needed for accurate assessment of the shape of the dose-response curve for radiationinduced health effects in the intact organism. Computational models can contribute to the improved understanding of mechanisms through integration of data and quantitative evaluation of hypotheses. We propose to develop a novel computational model of bystander effects elicited by oxidative stress and a conceptual basis for a “biological archetype.” The main components of the bystander effect model will be (a) a spatial grid, with each grid element containing a single cell, (b) a basal level of reactive oxygen species (ROS) in each cell with incremental levels due to ionizing radiation, (c) DNA damage due to ROS, (d) enzymatic repair of the damage, with a capability for evaluating induction of repair as an adaptive process linked to stress-related activation of intracellular signaling (e) diffusion between cells of ROS and components of the signaling pathway , (f) a cell cycle submodel that senses the amount of DNA damage and either holds the cell at a checkpoint, directs entry into the apoptotic pathway, or allows progression through the next stage of the cycle and (g) division of surviving cells to replace cells lost to apoptosis. Cells that progress through the cycle in the presence of radiation-induced DNA damage will have a proportionately increased probability of mutation. Background and radiation-induced oxidative stress in directly hit and bystander cells will thus be associated with a suite of possible outcomes including (1) no adverse effect, (2) DNA damage, (3) apoptosis, (4) cellular proliferation and (5) accumulation of mutations. The model will be parameterized against data to the greatest degree possible and will be capable of both posing and evaluating hypotheses about the development and consequences of bystander effects at the molecular, cellular, and tissue levels. Our proposal for a biological archetype will draw on our experience in developing computational models of whole-body pharmacokinetic mechanisms of environmental chemicals where key biological processes and structures are described in detail, while nonessential components are lumped together. The archetype will be capable of integrating mechanistic information across the relevant levels of biological organization to predict adverse health effects in intact organisms. We expect that the archetype will not be a single, large, complex model but rather a suite of models with due attention paid to programming standardization and capabilities for intermodel communication. Together, these efforts will demonstrate a rigorous computational modeling approach to the evaluation of hypotheses for mechanisms of bystander effects and contribute to development of a conceptual framework for the use of molecular level mechanistic data in human health risk assessment.

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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

2003 Abstract

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Title: Mechanisms of Low-Dose Inducible DNA Repair and the Adaptive Response

Authors: Janice Pluth, Hengameh Zahed Kargaran, Stacey Gauny, and Priscilla K. Cooper

Institutions: Life Sciences Division, Lawrence Berkeley National Laboratory

Neither the underlying mechanism nor the generality and extent of a protective effect to subsequent radiation-induced or endogenous damage (the radio-adaptive response) that can be induced by exposure to low level ionizing radiation (LLIR) is well understood at present. Such information is essential for development of meaningful models for assessing risk associated with exposure to low doses of ionizing radiation. Although DNA damage signaling and DNA repair processes appear likely to be involved, the detailed nature and regulation of repair processes responsive to LLIR is still largely unknown. Furthermore, important interconnections between the relevant repair pathways and with other essential DNA transactions -- including replication, transcription, chromatin dynamics, and cell cycle progression -- largely remain to be elucidated. While double-strand breaks (DSBs) are the most deleterious DNA damage produced by IR, a very much larger number of diverse base damages are directly produced, and these can be processed into DSBs either by replication or, when closely opposed, by lesion removal processes. An LLIR-inducible base excision repair (BER) process in human cells identified by M. Weinfeld (Le et al., Science 280:1066-1069, 1998) and shown to remove thymine glycols (Tg) more rapidly after a low priming dose of IR is thus a likely candidate for involvement in the adaptive response.

Preliminary studies by the Weinfeld group in collaboration with us have shown that human cells lacking transcription-coupled repair (TCR) because of mutations in the multi-functional repair protein XPG or in the Cockayne syndrome protein CSB do not exhibit LLIR-inducible BER. Our studies are aimed at understanding whether these and other proteins required for inducible BER are also critical for the adaptive response. We hypothesize that the salient damage induced both by LLIR and endogenous sources is oxidative base damage and strand breaks that, when encountered by a replication fork, result indirectly in double-strand breaks (DSBs). Further, we propose that recombinational repair coordinated with recruitment of the transcription-coupled base excision repair (TC-BER) machinery is required for removal of the lesion and restoration of fork progression. To test this hypothesis for the mechanism underlying the adaptive response, we have proposed to (1) characterize the roles of TCR-related proteins in responses to LLIR; (2) develop a highly sensitive assay to quantify the adaptive response; and (3) examine the possibility that TCR proteins are recruited to replication forks stalled by oxidative lesions. To date we have optimized a rapid and accurate FACS-based assay to quantitate histone ?H2AX as a measure of DSBs and have shown a linear (R2 =0.99) dose response relationship down to 10 cGy. We are currently working out conditions to accurately detect doses under 10 cGy as well. An adaptive response regimen of exposures is being tested on various normal and TCR-defective lines. Once conditions for an optimal adaptive response have been identified, the FACS-based assay will be used to detect differences in response between primed and naive cells after exposure to a challenge dose. We have also used immunofluorescence techniques to identify proteins that localize to stalled replication forks. We have observed that XPG co-localizes with Mre11 and NBS1, components of the MRN complex, which forms foci during S phase and in response to DNA damage. XPG foci formation is enhanced after exposure to hydroxyurea (HU), an agent known to cause stalled replication forks. These results imply that XPG may be important in resolving stalled replication forks. Our recent demonstration that XPG-defective cells are more sensitive to H2O2 than their complemented counterparts suggests that this function involves responses to oxidative damage, in agreement with our hypothesis for the mechanism underlying the adaptive response.

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