Profiles of global gene expression in ionizing-radiation-damaged human diploid fibroblasts reveal synchronization behind the G1 checkpoint in a G0-like state of quiescence.
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.
Department 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, NC 27599, USA.
Tong
T
Zhou
Jeff W
JW
Chou
Dennis A
DA
Simpson
Yingchun
Y
Zhou
Thomas E
TE
Mullen
Margarida
M
Medeiros
Pierre R
PR
Bushel
Richard S
RS
Paules
Xuebin
X
Yang
Patrick
P
Hurban
Edward K
EK
Lobenhofer
William K
WK
Kaufmann
D002460:Cell Line; D004249:DNA Damage; D004171:Diploidy; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000166:cytology / Q000528:radiation effects*; D016193:G1 Phase; D020869:Gene Expression Profiling; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011839:Radiation, Ionizing; D016192:Resting Phase, Cell Cycle