Abstract
Embryonic stem (ES) cells, and the inner cell mass from which they are derived, are hypersensitive to DNA damage and appear to have specific cellular defense systems for DNA repair and the elimination of damaged cells. These mechanisms differ from somatic cells and are vital to minimize developmental defects that would potentially result from the continued proliferation and differentiation of abnormal cells into adult cell lineages. Although the DNA damage-induced signaling cascades activated in these cells are known to include p38 and c-Jun N-terminal protein kinase mitogen-activated protein kinase pathways and activation of a variety of transcription factors, including p53, nuclear factor-êB, and activator protein-1, the nature of the specific mechanisms unique to these cells remains to be elucidated. Here, we describe the use of homozygous knockout ES cells to investigate the role of Ets1 in the response to DNA damage in these cells. These studies demonstrate that Ets1 is required for optimal p53 function in this response and further demonstrate the potential for knockout ES cells to elucidate the role of specific genes in early embryonic cell responses.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Heyer B. S., MacAuley A., Behrendtsen O., and Werb Z. (2000) Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development. Genes Dev. 14, 2072–2084.
Hardy K., Handyside A. H., and Winston R. M. (1989) The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 107, 597–604.
Thomas K. R. and Capecchi M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.
Evan G. and Littlewood T. (1998) A matter of life and cell death. Science 281, 1317–1322.
Corbet S. W., Clarke A. R., Gledhill S., and Wyllie A. H. (1999) P53-dependent and-independent links between DNA-damage, apoptosis and mutation frequency in ES cells. Oncogene 18, 1537–1544.
de Gruijl F. R., van Kranen H. J., and Mullenders L. H. (2001) UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer. J. Photochem. Photobiol. B 63, 19–27.
Hanawalt P. C. (2002) Subpathways of nucleotide excision repair and their regulation. Oncogene 21, 8949–8956.
Zhou B. B. and Elledge S. J. (2000) The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.
Bender K., Blattner C., Knebel A., Iordanov M., Herrlich P. and Rahmsdorf H. J. (1997) UV-induced signal transduction. J. Photochem. Photobiol. B 37, 1–17.
Hwang S. Y., Hertzog P. J., Holland K. A., et al. (1995) A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons α and β and alters macrophage responses. Proc. Natl. Acad. Sci. USA 92, 11,284–11,288.
Lahoud M. H., Ristevski S., Venter D. J., et al. (2001) Gene targeting of Desrt, a novel ARID class DNA-binding protein, causes growth retardation and abnormal development of reproductive organs. Genome Res. 11, 1327–1334.
Tessarollo L. (2001) Manipulating mouse embryonic stem cells. Methods Mol. Biol. 158, 47–63.
Xu D., Wilson T. J., Chan D., et al. (2002) Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells. EMBO J. 21, 4081–4093.
Roberts K. M., Rosen A., and Casciola-Rosen L. A. (2004) Methods for inducing apoptosis. Methods Mol. Med. 102, 115–128.
Gong J., Traganos F., and Darzynkiewicz Z. (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem. 218, 314–319.
Zauberman A., Lupo A., and Oren M. (1995) Identification of p53 target genes through immune selection of genomic DNA: the cyclin G gene contains two distinct p53 binding sites. Oncogene 10, 2361–2366.
Zauberman A., Flusberg D., Haupt Y., Barak Y., and Oren M. (1995) A functional p53-responsive intronic promoter is contained within the human mdm2 gene. Nucleic Acids Res. 23, 2584–2592.
Owczarek C. M., Hwang S. Y., Holland K. A., et al. (1997) Cloning and characterization of soluble and transmembrane isoforms of a novel component of the murine type I interferon receptor, IFNAR 2. J. Biol. Chem. 272, 23,865–23,870.
Sabapathy K., Klemm M., Jaenisch R., and Wagner E. F. (1997) Regulation of ES cell differentiation by functional and conformational modulation of p53. EMBO J. 16, 6217–6229.
Wobus A. M., Holzhausen H., Jakel P., and Schoneich J. (1984) Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res. 152, 212–219.
Chao C., Saito S., Kang J., Anderson C. W., Appella E., and Xu Y. (2000) p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage. EMBO J. 19, 4967–4975.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Xu, D., Wilson, T.J., Hertzog, P.J. (2006). Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 329. Humana Press. https://doi.org/10.1385/1-59745-037-5:327
Download citation
DOI: https://doi.org/10.1385/1-59745-037-5:327
Publisher Name: Humana Press
Print ISBN: 978-1-58829-498-2
Online ISBN: 978-1-59745-037-9
eBook Packages: Springer Protocols