doi:10.1016/j.yexcr.2004.10.036 
Copyright © 2004 Elsevier Inc. All rights reserved.
Early BrdU-responsive genes constitute a novel class of senescence-associated genes in human cells
Sachi Minagawaa, Kazuhiko Nakabayashib, Michihiko Fujiia, Stephen W. Schererb and Dai Ayusawaa, 
, 
aKihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Yokohama 244-0813, Japan
bGenetics and Genomic Biology Program, The Hospital for Sick Children and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Received 12 July 2004;
revised 21 October 2004.
Available online 15 December 2004.
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Abstract
We identified genes that immediately respond to 5-bromodeoxyuridine (BrdU) in SUSM-1, an immortal fibroblastic line, with DNA microarray and Northern blot analysis. At least 29 genes were found to alter gene expression greater than twice more or less than controls within 36 h after addition of BrdU. They took several different expression patterns upon addition of BrdU, and the majority showed a significant alteration within 12 h. When compared among SUSM-1, HeLa, and TIG-7 normal human fibroblasts, 19 genes behaved similarly upon addition of BrdU. In addition, 14 genes, 9 of which are novel as regards senescence, behaved similarly in senescent TIG-7 cells. The genes do not seem to have a role in proliferation or cell cycle progression. These results suggest that the early BrdU-responsive genes represent early signs of cellular senescence and can be its new biomarkers.
Keywords: 5-Bromodeoxyuridine; Microarray; Gene expression; Cellular senescence; Biomarker
Fig. 1. Northern blot analysis of the early BrdU-responsive genes in SUSM-1. Total mRNA samples were prepared from the cells cultured in the presence of BrdU for the time indicated. Northern blot analysis was performed with cDNA probes for the genes indicated as described in Materials and methods.
Fig. 2. Gene expression patterns of the early BrdU-responsive genes in SUSM-1. (A) Clustering analysis of time-dependent changes in mRNA levels upon addition of BrdU. After addition of BrdU to the cells, mRNA levels for each gene were determined at intervals by Northern blot analysis as described in Materials and methods. Symbols (A–I) correspond to the patterns shown in the right panels. Vertical bar indicates that the gene behaved similarly in senescent TIG-7 cells. (B) Expression patterns of the genes. After clustering analysis of the mRNA levels for each gene, nine different expression patterns (A–I) were obtained as illustrated.
Fig. 3. Venn diagram of the early BrdU-responsive genes in SUSM1, HeLa, and TIG-7. The 29 early BrdU-responsive genes identified in SUSM-1 were assigned to overlapping regions of the Venn diagram. Numbers in parentheses indicate that the genes behaved similarly in senescent TIG-7 cells.
Table 1.
Early BrdU-responsive genes showing expression greater than twice more or less than control upon addition of BrdU in SUSM-1 cells
a The genes marked with asterisks are already described as regards cellular senescence.
Table 2.
Time courses of expression of early BrdU-responsive genes after addition of BrdU in SUSM-1 cells
a The genes underlined behaved similarly in senescent TIG-7 cells.
Table 3.
Expression profiles of early BrdU-responsive genes in SUSM-1, HeLa, and TIG-7 upon addition of BrdU
Asterisks and underlines are as in Table 1 and Table 2.
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