| Solid tumors comprise genetically heterogeneous cell
populations whose growth and survival depend upon
the complex interplay of distinct, yet overlapping,
signaling networks. A major challenge in developing a
course of therapy is determining which signaling
nodes to target for a specific malignancy. Profiles
from siRNA gene silencing are integral to mapping
disease-specific signaling cascade(s) and provide
insight to key targets for therapeutic intervention.
Successful siRNA screening relies not solely upon
optimizing transfection, but also cell analysis systems
capable of high content screening (HCS) at the single
cell level, within overall populations (sample well),
and across multiple data sets. The Guava EasyCyte™
Plus flow cytometer, with integrated Guava® Simplicity
software, provides a revolutionary new platform for
cell-based analysis. The software’s intuitive
architecture and ease of use facilitates the comparison
of multiple experimental conditions or disease states
through heat-map visualization. To demonstrate, a
mini-drug screening was carried out. Following
exposure to a panel of 80 cytoactive compounds, cells
were assayed for multiple parameters of apoptotic
induction as well as monitoring alterations in mitotic
state. Parallel to screening, siRNA silencing was
performed to identify genes that impact Camptothecin
(CPT)-induced apoptosis. Knockdown efficiency at
each gene target was examined via intracellular
staining and optimized for each cell line prior to
functional screening. “Hit” compounds were further
tested in combination with siRNA knockdown. For this
study, changes in the phosphorylation state of
multiple proteins were also measured. Briefly,
apoptotic assays following gene silencing identified
enhancers (PTEN) and inhibitors (GSK3a) of CAMinduced
cell death as well as modulators of cell cycle
progression (MDM2, CHK1). Moreover, it was evident
that certain genes are functionally linked and thus
part of the same or overlapping networks. “Hit”
screening identified cytochalasin as a potential
therapeutic with similar biological profiles as CPT.
However, phospho-mapping suggest their specific
mechanisms of action are quite different. In
summary, this experimental methodology, when used
in concert with Guava Technologies’ cell analysis
platform and Simplicity software, significantly
expedites the drug discovery process by providing a
means for extraction of key biological findings from
complex experimental results. |
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