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Show SCHOOL OF MEDICINE AND HEALTH SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS Kallie Bracken Nikolaus Trede 110 DEVELOPING A HIGH THROUGHPUT PROTOCOLTO MEASURE INHIBITION OF GROWTH FACTOR REGULATED PATHWAYS IN HEMATOPOIETIC CELLS Kallie Bracken (Nikolaus Trede) Departments of Pediatrics and Oncological Sciences University of Utah The Trede Lab focuses on research to discover and develop drugs to treat T-cell acute lymphoblastic leukemia (T-ALL).The In-Cell Western (ICW) assay, if modified to keep important growth pathways active during incubation with drug treatments, could provide a n e w way to analyze drug candidates, or to perform structure activity relationship (SAR) studies. To keep the growth pathways active, the growth serum, which unfortunately causes the cells to stick very loosely to the plate, must be included during incubation. Therefore the challenge is to adjust the protocol, originally designed by LI-COR Biosciences, to minimize loss of cells due to the inclusion of the serum. Keeping important growth pathways active makes it possible through the ICW to detect the activity of drug treatments that inhibit the pathways. Once published, this data will provide a novel technique for measuring growth factor regulated pathways. The ICW assay is an immunocytochemical assay performed in a microplate. Cells are plated and treated with specific inhibitors or drugs and treated with target-specific primary antibodies as well as secondary antibodies that can be detected as a fluorescent signal. Their fluorescence provides a way to quantify the presence of the target protein, and therefore to see the effects of the original drugs or inhibitors on the target. The main benefit of ICW assays is that they provide a quick and accurate way to detect proteins and therefore analyze the effects of drugs in a cellular context. One key growth factor regulated pathway involves the protein AKT, which plays a vital role in many cellular processes such as apoptosis and proliferation. Because AKT drives cell proliferation, it is implicated with many cancers; treatments that interact with AKT to stop or hinder the growth of cells are very significant in the treatment of m a n y hematopoietic malignancies, including T-ALL. SAR studies, which can be performed though ICW assays, can be used to develop drug candidates and discover h o w they might be affecting AKT downstream pathways. Once again, for this to be possible using the ICW, pathways must be kept active by including the growth serum before incubation. In contrast, LI-COR's original protocol removes the serum which can reduce the signal from these pathways, minimizing the signal window to detect inhibitory effects. Perfecting the ICW protocol is a step closer to understanding the mechanism behind drugs such as LDK, a compound discovered by the Trede Lab for its ability to eliminate malignant T-cells. Furthermore, the ICW provides a high throughput method for drug screening as well as SAR studies using suspension cells. References LI-COR Biosciences. In-Cell Western Assay. LI-COR Biosciences USA. Webpage: http://www.licor.com/bio/applications/odys-sey_ applications/in_cell_western.jsp Zhang, J.H., Chung, T.D., Oldenburg, K.R.. A Simple Statistical Parameter fo Use in Evaluation and Validation Throughput Screening Assays. J Biomol Screen. 1999; 4(2): 67-73 |
