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Cell-Based Assays for High-Throughput Screening pp 139–149Cite as

Assay for Isolation of Inhibitors of Her2-Kinase Expression

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 486))

Summary

Her2 (ErbB2) protein is overexpressed in breast and other solid tumors, and its expression is associated with progressive disease. Current therapies directed toward Her2 either block dimerization of the receptor or inhibit tyrosine kinase activity to disrupt intracellular signaling. However, little is known about alternative mechanisms for suppressing Her2 expression, possibly by inducing degradation or blocking synthesis. Here, we describe a hybrid western-blotting and enzyme-linked immunosorbent assay (ELISA) designed to identify in low- to medium-throughput format noncytotoxic compounds that reduce expression of Her2 protein.

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References

  1. Yarden, Y. and Sliwkowski, M. X. (2001) Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol. 2, 127–137.

    Article  PubMed  CAS  Google Scholar 

  2. Klapper, L. N., Kirschbaum, M. H., Sela, M., and Yarden, Y. (2000) Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv. Cancer Res. 77, 25–79.

    Article  PubMed  CAS  Google Scholar 

  3. Yu, D. and Hung, M. C. (2000) Over-expression of ErbB2 in cancer and ErbB2-targeting strategies. Oncogene 19, 6115–6121.

    Article  PubMed  CAS  Google Scholar 

  4. Ross, J. S. and Gray, G. S. (2003) Targeted therapy for cancer: the HER-2/neu and Herceptin story. Clin. Leadersh. Manag. Rev. 17, 333–340.

    PubMed  Google Scholar 

  5. Arteaga, C. (2003) Targeting HER1/EGFR: a molecular approach to cancer therapy. Semin. Oncol. 3 Suppl 7, 3–14.

    Article  Google Scholar 

  6. Citri, A., Gan, J., Mosesson, Y., Vereb, G., Szollosi, J., and Yarden, Y. (2004) Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation. EMBO Rep. 12, 1165–1170.

    Article  Google Scholar 

  7. Citri, A., Kochupurakkal, B. S., and Yarden, Y. (2004) The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention. Cell Cycle 1, 51–60.

    Google Scholar 

  8. Aoyagi, S. and Archer, T. K. (2005) Modulating molecular chaperone Hsp90 functions through reversible acetylation. Trends. Cell Biol. 11, 565–567.

    Article  Google Scholar 

  9. Huezo, H., Vilenchik, M., Rosen, N., and Chiosis, G. (2003) Microtiter cell-based assay for the detection of agents that alter cellular levels of Her2 and EGFR. Chem. Biol. 10, 629–634.

    Article  PubMed  CAS  Google Scholar 

  10. Llauger, L., He, H., Kim, J., Aguirre, J., Rosen, N., Peters, U., Davies, P., and Chiosis, G. (2005) 8-Arylsulfanyl and 8-arylsulfoxyl adenine derivatives as inhibitors of the heat shock protein 90. J. Med. Chem. 48, 2892–2905.

    Article  PubMed  CAS  Google Scholar 

  11. He, H., Zatorska, D., Kim, J., Aguirre, J., Llauger, L., She, Y., Wu, N., Immormino, R. M., Gewirth, D. T., and Chiosis, G. (2006) Identification of potent water-soluble purine-scaffold inhibitors of the heat shock protein 90. J. Med. Chem. 49, 381–390.

    Article  PubMed  CAS  Google Scholar 

  12. Stockwell, B. R., Haggarty, S. J., and Schreiber, S. L. (1999) High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications. Chem. Biol. 2, 71–83.

    Article  Google Scholar 

  13. Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R. W., Schreiber, S. L., and Mitchison, T. J. (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974.

    Article  PubMed  CAS  Google Scholar 

  14. Haggarty, S. J., Mayer, T. U., Miyamoto, D. T., Fathi, R., King, R. W., Mitchison, T. J., and Schreiber, S. L. (2000) Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis. Chem. Biol. 4, 275–286.

    Article  Google Scholar 

  15. Blaskovich, M. A., Sun, J., Cantor, A., Turkson, J., Jove, R., and Sebti, S. M. (2003) Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Res. 63, 1270–1279.

    PubMed  CAS  Google Scholar 

  16. Haggarty, S. J., Koeller, K. M., Wong, J. C., Grozinger, C. M., and Schreiber, S. L. (2003) Domain-selective small molecule inhibitor of HDAC6-mediated tubulin deacetylation. Proc. Natl. Acad. Sci. USA 100, 4389–4394.

    Article  PubMed  CAS  Google Scholar 

  17. Haggarty, S. J., Koeller, K. M., Kau, T. R., Silver, P. A., Roberge, M., and Schreiber, S. L. (2003) Small molecule modulation of the human chromatid decatenation checkpoint. Chem. Biol. 12, 1267–1279

    Article  Google Scholar 

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Acknowledgments

The author would like to thank Joungnam Kim and Cristina C. Clement for technical assistance. This work was supported by R03 MH076408, and a Susan G. Komen grant, R01-SGK-BCTR0504381.

Author information

Authors and Affiliations

  1. Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Gabriela Chiosis

  2. Southern Research Molecular Libraries Screening Center, Assay Implementation Group, Southern Research Institute, Birmingham, AL, USA

    Adam B. Keeton

Authors
  1. Gabriela Chiosis
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  2. Adam B. Keeton
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Editor information

Editors and Affiliations

  1. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Paul A. Clemons

  2. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Nicola J. Tolliday

  3. Broad Institute of Harvard, Chemical Biology Program, Massachusetts Institute of Technology, Cambridge Center 7, Cambridge, 02142, U.S.A.

    Bridget K. Wagner

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Chiosis, G., Keeton, A.B. (2009). Assay for Isolation of Inhibitors of Her2-Kinase Expression. In: Clemons, P., Tolliday, N., Wagner, B. (eds) Cell-Based Assays for High-Throughput Screening. Methods in Molecular Biology, vol 486. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-545-3_10

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  • DOI: https://doi.org/10.1007/978-1-60327-545-3_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-544-6

  • Online ISBN: 978-1-60327-545-3

  • eBook Packages: Springer Protocols

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