OBJECTIVES

Core 1. Signal Transduction Rapid Assessment Core

The Luminex technology has been successfully developed for multiplexed analysis of cytokines. It is the goal of this proposal to identify monoclonal antibodies that can detect the activation-state of the signal transduction cascade as it specifically applies to breast cancer. The long-term goal is to develop this technology so that it can be applied to the rapid assessment of activation-state of signal transduction in primary tumor samples. Specifically this will involve:

1. Detection of phsopho-tyrosine signaling in breast cancer. Monoclonal antibodies against proteins whose activation state is heralded by the presence of phsopho-tyrosine will be evaluated. In particular, antibodies against receptor and non-receptor tyrosine kinases as well as downstream signaling molecules will be assessed on the Luminex platform with an anti-phsopho-tyrosine antibody (4G10 or related antibodies).
   
   
2. Screen pairs of antibodies for detection of activated signaling in breast cancer. Signaling molecules become activated by phosphorylation of a specific residue. Pairs of antibodies that capture a signaling molecule and recognize a specific phospho-residue will be evaluated on the Luminex system.

James A. DeCaprio, M.D.-Dr. DeCaprio is a physician-scientist and medical onclogist with a primary interest in the function of oncogenes. He is an Associate Professor of Medicine, Dana-Farber Cancer Institute and Harvard Medical School. He is an expert in the development of activation specific monoclonal antibodies. He will direct the Rapid Signal Assessment Core (Core 1). Email: James_DeCaprio@dfci.harvard.edu

Thomas M. Roberts, Ph.D.-Dr. Roberts is Professor of Pathology, Dana-Farber Cancer Institute and Harvard Medical School. He is a leader in the signal transduction field and the role of PI kinase pathways in cancer. He will serve as a co-investigator on Project 3 and a collaborator on Core 1. Email: Thomas_Roberts@dfci.harvard.edu

The Luminex¹⁰⁰ system utilizes as many as 50 different colored microspheres that can be coated with a unique antibody. The beads are mixed with the appropriate protein sample and then a secondary antibody labeled with a fluorescent probe that can also recognize the captured antigen is added. The Luminex device then detects the relative level of secondary antibody for each different colored microsphere. Using this technology, a rapid and simultaneously assessment of 15 cytokines has been reported.

Preliminary data has confirmed that a specific monoclonal antibody (LA1, Upstate Biotechnology) against the EGF1 receptor (EGF1-R) can be formatted on the Luminex platform (T. Roberts, data not shown). Using as little as 4 ug of lysate derived from HeLa cells stimulated with EGF, the presence of activated EGF1-R could be specifically detected by a phospho-tyrosine specific monoclonal antibody 4G10. Given this, it is conceivable that as many as 50 different signaling molecules can be simultaneously assessed for the degree of phospho-tyrosine.

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