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Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop V

2005 Abstract

Title: Gene Expression in Thyroid Tissue Analogs Following Acute Low Dose and Low Dose Rate Gamma Irradiation

Authors: LM Green, BM Bianski, LS Ortloff & E Kyeyune-Nyombi, Radiobiology Program, Chan Shun Pavilion room A1010 Loma Linda, CA 92354 E-mail lgreen@dominion.llumc.edu

INTRODUCTION: The thyroid, like most epithelial glands are a common site of tumor development and sensitive to ionizing radiation induced carcinogenic changes. We have characterized a thyroid cell line that has been used as our model for investigating radiation effects [1-4], however, in order to better assess normal tissue properties we established thyroid tissue analogs in vitro that better approximates the conditions in vivo. The thyroid tissue analogs produced under reduced gravitational forces dramatically affects differentiation and growth patterns facilitating three-dimensional assembly of the thyrocytes into multifollicular structures [5]. Cells grown in the low-shear stress rotating-wall vessels allow us to evaluate various aspects of complex interactions that are not possible using traditional culture techniques. Thus, our model provides us the opportunity to investigate how a relatively normal tissue responds to ionizing radiation at multiple levels. The goal of the current project was to characterize the pattern of radiation modulated gene expression in thyroid tissue functional units (FTU’s) using low-dose/low-dose rate ionizing radiation.

This presentation will details our findings, thus far, including the results of differentially expressed gene products in response to acute and protracted exposure to low dose gamma radiation. Further the low dose rate primed tissues were challenged with an acute exposure of gamma-rays and assessed for differences in response compared to equal doses delivered acutely. Research Design & Results--Gene expression patterns from the acute exposures were evaluated by several criteria including comparing the basal levels of gene expression in the 0 cGy 3hrs, 2 and 7 day controls to 10, 20 and 50 cGy gamma-rays. Comparative analysis was conducted to measure differences between dose and time parameters. In addition to global analysis, gene profiles were analyzed using GeneSpring software (Silicon Genetics). The selection procedure screened first by significance (p<0.05), then by presence across replicates and then by fold-change (modulated > 1.5 fold compared to basal levels). A minimum of duplicate microarrays consisting of 15, 900 rat genes were processed for each condition and appropriate controls.

The resulting gene expression differences were greatest in the number of modulated genes (>1.5 fold) for the 3 hour samples for all doses, whereas, the 2 day time point was reduced in number. The number of modulated genes then increased in nearly all categories at 7 days post exposure (with the exception of the 20 cGy samples). The majority of modulated genes fell into categories of cell cycle related proteins, epithelial and extracellular matrix related proteins, oncogenes, signal transduction components (kinases) and thyroid related products.

The low-dose rate exposures were conducted similarly with a minimum of duplicates harvested for microarray analysis following a 2 day protracted exposure. Samples were harvested immediately following the protracted irradiation, 3 hours and 2 days post exposure. Additional samples were challenged with an acute 10 cGy exposure of gamma-rays after the low-dose rate exposure was completed, with samples harvested at 3hours and 2 days later. The comparative results revealed that the low dose rate exposed tissue analogs had an increased response in the magnitude and number of modulated genes compared to the samples exposed to the equivalent dose delivered acutely. Additionally, when the low dose rate samples were
challenged with an equivalent acute dose of gamma-rays the response was decreased relative to the controls (see Table 1).

Table 1
Gene Expression Data for Low Dose Rate Exposures
Plus/Minus Secondary Challenge Exposures

Conclusions—The response of the acute exposure experiments suggests that the initial response perturbed a number of immediate response genes that later subsided (by 48 hours), followed by a new round of increased expression at 7 days. Interestingly, there was reciprocallity to the genes modulated; in general, the genes that were elevated at 3 hours were the same genes that were decreased at 7 days. The reverses also appeared to be true, in that, the genes decreased to the lowest levels at 3 hours were the genes increased to the greatest level in the 7 day samples. When the gene expression patterns were compared between the acute and protracted irradiated tissue analogs (shown above); it appeared that the low-dose-rate exposure resulted in a increased response which was dampened when the tissue analogs received an acute challenge exposure of equivalent dose. This suggests that the low dose rate priming had a protective effect relative to the secondary challenge exposure. The identities of the specific differential genes expressed under these conditions are being investigated.

References

1. Green LM, Murray D, Tran D, Bant A, Kazarians G, Moyers M & Nelson G. Radiat Res 155- 32-42. (2001)
   
   
2. Green LM, Murray DK, Tran DT, Nelson GA, Shah MA & Luben RA. J. Mol Endocrinol 27(2): 145-163 (2001)
   
   
3. Green LM, Tran DT, Murray DK, Rightnar SS, Todd S, & Nelson GA. Radiat. Res. 158:475-485, (2002)
   
   
4. Green LM, Bianski B, Murray DK, Rightnar SS & Nelson GA. Radiat. Res. 163:172-182 (2005)
   
   
5. Green LM, Patel Z, Murray D, Rightnar S, Burrell C, Gridley DS & Nelson GA, J Radiat. Res.43:213-218, (2002)

Acknowledgements This research was supported by DOE Grant DE-FG02-02ER63448 and a NASA grant NAG 9-1452.