Abstract
To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle. When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase. Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells.
Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase. Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1. Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.
In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics. In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization.
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Nyberg, K. A., Michelson, R. J., Putnam, C. W, and Weinert, T. A. (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36, 617–656.
Visintin, R., Prinz, S, and Amon, A. (1997) CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 278, 460–463.
Sigrist, S. J. and Lehner, C. F. (1997) Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90, 671–681.
Schwab, M., Lutum, A. S, and Seufert, W. (1997) Yeast Hct1 is a regulator of Clb2 cyclin proteolysis. Cell 90, 683–693.
Sudo, T., Ota, Y., Kotani, S., et al. (2001) Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells. EMBO J. 20, 6499–6508.
Musacchio, A. and Hardwick, K. G. (2002) The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell Biol. 3, 731–741.
Polyak, K., Kato, J. Y., Solomon, M. J., et al. (1994) p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9–22.
Nourse, J., Firpo, E., Flanagan, W. M., et al. (1994) Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372, 570–573.
Davis, F. M., Tsao, T. Y., Fowler, S. K, and Rao, P. N. (1983) Monoclonal antibodies to mitotic cells. Proc. Natl. Acad. Sci. USA 80, 2926–2930.
Rieder, C. L. and Palazzo, R. E. (1992) Colcemid and the mitotic cycle. J. Cell Sci. 102(Pt 3), 387–392.
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© 2004 Humana Press Inc.
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Sudo, T., Ueno, N.T., Saya, H. (2004). Functional Analysis of APC-Cdh1. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology, vol 281. Humana Press. https://doi.org/10.1385/1-59259-811-0:189
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DOI: https://doi.org/10.1385/1-59259-811-0:189
Publisher Name: Humana Press
Print ISBN: 978-1-58829-500-2
Online ISBN: 978-1-59259-811-3
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