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Tong Zhou,¹ Jeff W. Chou,² Dennis A. Simpson,¹ Yingchun Zhou,¹ Thomas E. Mullen,¹ Margarida Medeiros,¹ Pierre R. Bushel,² Richard S. Paules,² Xuebin Yang,¹ Patrick Hurban,³ Edward K. Lobenhofer,³ and William K. Kaufmann¹

¹Department of Pathology and Laboratory Medicine, Center for Environmental Health and Susceptibility, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; ²National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA; ³Paradigm Array Labs, Icoria Inc., Research Triangle Park, North Carolina, USA

Abstract
Cell cycle arrest and stereotypic transcriptional responses to DNA damage induced by ionizing radiation (IR) were quantified in telomerase-expressing human diploid fibroblasts. Analysis of cytotoxicity demonstrated that 1.5 Gy IR inactivated colony formation by 40-45% in three fibroblast lines ; this dose was used in all subsequent analyses. Fibroblasts exhibited > 90% arrest of progression from G₂ to M at 2 hr post-IR and a similarly severe arrest of progression from G₁ to S at 6 and 12 hr post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 hr post-IR caused the S and M compartments to empty by > 70% at 24 hr. Global gene expression was analyzed in IR-treated cells. A microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression that were common to the three fibroblast lines, including a dominant p53-dependent G₁ checkpoint response. Many p53 target genes, such as CDKN1A, GADD45, BTG2, and PLK3, were significantly up-regulated at 2 hr post-IR. Many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 hr post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts apparently as a result of cell synchronization behind the G₁ checkpoint. However, cluster and principal component analyses of gene expression revealed a profile 24 hr post-IR with similarity to that of G₀ growth quiescence. The results reveal a highly stereotypic pattern of response to IR in human diploid fibroblasts that reflects primarily synchronization behind the G₁ checkpoint but with prominent induction of additional markers of G₀ quiescence such as GAS1. Key words: cell cycle checkpoints, DNA damage, gene expression, human fibroblasts, ionizing radiation, microarray. Environ Health Perspect 114: 553-559 (2006) . doi:10.1289/ehp.8026 available via http://dx.doi.org/ [Online 19 December 2005]

Address correspondence to W.K. Kaufmann, CB#7295, Room 31-325, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295 USA. Telephone: (919) 966-8209. Fax: (919) 966-9673. E-mail: wkarlk@med.unc.edu

We thank J. Helms for performing tests for mycoplasma contamination, Y. Shi and R. Tian for help with pilot microarray experiments, and G. Wu, D. Xiang, and J. Li for help with microarray data analysis. We acknowledge the U.S. Public Health Service's contribution of the software Expression Analysis Systematic Explorer (EASE) used in our data analysis.

This project was funded with federal funds from the National Institute of Environmental Health Sciences, National Institutes of Health, under contract N01-ES-25497, and U.S. Public Health Service (PHS) grants ES11391 and ES10126.

P.H. and E.K.L. are employed by Icoria, Inc. The remaining authors declare they have no competing financial interests.

Received 17 February 2005 ; accepted 15 December 2005.