PNNL-13501 - Pacific Northwest National Laboratory

Characterization of Nodes in Cell-Signaling Pathways Using High-Throughput
Array Technologies
David L. Springer, Kwong Kwok Wong, Richard C. Zangar, Susan M. Varnum, John H. Miller,
Catherine E. Petersen, Karen L. Wahl, Karin D. Rodland
Study Control Number: PN99007/13335
This project is developing new technologies to address broad issues of gene expression and protomics. Our ultimate goal
is to understand cell signaling pathways and networks and determine how they are regulated. Identification and
characterization of proteins involved in cell signaling events and how they are altered by environmental exposures will
provide critical information regarding the onset and progression of human disease.
Project Description
Scientific studies of gene expression and proteomics use
information from cDNA arrays to identify genes whose
expression is altered by environmental stresses and
disease states. In addition, antibody arrays are being
developed to selectively and quantitatively capture
specific proteins from cellular preparations. Once
captured, the proteins are identified using advanced mass
spectrometric techniques, and their post-translational
modification status is determined. Changes in cellular
proteins will then be related to cell function. These new
approaches will enhance our understanding of how
changes in the proteome in response to environmental
agents contribute to the onset and progression of human
diseases.
Introduction
To better understand the disease process, it is necessary to
understand both normal and abnormal signaling. A major
function of proliferation-associated signal transduction
pathways is regulation of the cell’s decision to proliferate
or not, based on the integration of numerous extracellular
(and therefore environmental) stimuli. Among those
extracellular molecules capable of modifying cellular
behavior, calcium and other multivalent cations including
cadmium and gadolinium have recently been identified as
key mediators of the balance between proliferation and
differentiation. In mammalian cells, a recently cloned Gprotein
coupled receptor, the calcium-sensing receptor
(CaR), has been shown to be the key player in sensing
changes in these small molecules and translating them
into proliferative responses. One disease in which the
CaR appears to play an essential role is ovarian cancer,
and we have selected this disease as a test bed for
48 FY 2000 Laboratory Directed Research and Development Annual Report
development of new high-throughput technologies.
Professor Rodland’s laboratory at Oregon Health Sciences
University, Portland, has demonstrated that the cell type
responsible for human ovarian cancer, the ovarian surface
epithelial cell, is critically dependent on relatively high
levels of extracellular calcium for proliferation, and the
CaR is the molecule responsible for initiating this
response (McNeil et al. 1998; Hobson et al. 2000). The
presence of an aberrant truncated version of the CaR in
75% of highly malignant ovarian tumors (stages III and
IV) was characterized by Rodland. The major focus of
this project is to delineate the precise pathways by which
normal CaR modulates the proliferation of ovarian
surface epithelial cells to define how these pathways are
disrupted by the aberrant receptor in the process of
ovarian carcinogenesis.
Approach
Traditional approaches to the study of signaling pathways,
such as those based on co-immunoprecipitation, affinity
chromatography, 2D-PAGE, and in vitro kinase assays,
have established that signaling pathways involve the
concerted interaction of multiple proteins in series and in
parallel, and that these interactions most frequently
involve specific post-translational modifications which
serve as the activating step for the next round of
signaling. Despite the considerable advances in
knowledge provided by these older technologies, they
have serious shortcomings that limit our ability to fully
comprehend signaling processes. Traditional biochemical
approaches are limited to interactions between known
proteins with high affinity for each other and that are
present at relatively abundant levels. In this project, we
are applying state-of-the-art technologies dependent on
antibody technology coupled to mass spectroscopy to