) Microfabricated fluidics devices originally designed for the Human Genome Project will be developed as a means to screen single tumor cells for aberrantly regulated kinase activity. Kinases regulate nearly all cellular functions including those critical for oncogenesis. The long-term goal of this work is to determine the number, identities, and degree of activation of kinases in individual cells within a malignancy. This information will aid kinase antagonist development, enhance patient prognosis, and identify potential therapeutic regimens. The investigators have established a collaboration to combine their respective talents to develop this new technology using a concerted, stepwise approach. The Allbritton Lab is highly experienced with the use of capillary electrophoresis for single-cell measurements of signal transduction molecules and kinase activity, and the Ramsey Lab has pioneered the development of these """"""""lab-on-a-chip"""""""" microfabricated devices. The devices to be developed will measure the activity of multiple kinases in a single tumor cell by clectrophoretic identification and quantification of phosphorylated and nonphosphorylated fluorescently-labeled, kinase-substrate peptides. This general approach will be applicable to single-cell measurements of the activities of a broad range of enzymes. The microchip format has the capability for single-cell measurements by virtue of exquisite sensitivity, extemely high separation efficiency, and miniaturization. This chip-based approach also lends itself to a contained, monolithic, cost-effective device for widespread dissemination to research and clinical labs alike.
The specific aims of this proposal are directed at method development and validation for physiologic measurements in single cells.
These aims are to define the separation conditions and detection limits for the substrate peptides on the chip, to optimize the cellular manipulations needed for the measurement, to determine the biologic properties of the peptide indicators, and to measure the activities of multiple kinases in a single tumor cell. When fully developed, this integrated microchip will represent a new paradigm in single-cell measurement techniques.
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