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P8003
High-throughput DNA Damage Assay for Breast Cancer Related Protein Markers using the IsoCyte™ Platform
Presenter Evan Cromwell, Blueshift Biotechnologies, USA
Additional Authors: Brian T. Bennett, Madeline Craske, Jorg Bewersdorf, Kendall L. Knight, Steven C. Miller
Breast cancer in women between the ages of 40 – 55 is the leading cause of death in the United States each year and the second leading cause of death for all women.  Treatment regimens range from surgery (lumpectomy or mastectomy) to chemotherapy, use of anti-cancer drugs and ionizing radiation to drug therapy.  Important to our understanding of how these treatments affect cancerous cells is an understanding of the complicated molecular events surrounding breast cancer related protein markers such as phosphorylated Histone H2AX, which is the accepted marker for a DNA double strand break, Breast Cancer Related Protein 2 (BRCA2) and other breast cancer related proteins.  It has been reported and accepted that in response to breast cancer chemotherapeutics and ionizing radiation that gamma-H2AX, BRCA2 and other breast cancer related proteins localize to the nucleus and form nuclear foci.  To date foci formation and timing have been analyzed and quantified using traditional microcopy techniques requiring the production of many samples and counting of individual cells within a field or fields of cells.  However, this technique can be flawed by the individual interpretation of the protein localization as well as the ability to collect statistically relevant numbers.  Here we employ the IsoCyte technology, a high-throughput laser scanning system, to undertake the first examination of large human cell populations with respect to breast cancer associated proteins by this method.  The timing of their response, cellular localization, foci appearance and clearance after drug treatment will all be simultaneously measured.   We report the examination of multiple human cell lines, counting on the order of hundreds of thousands of cells, while simultaneously collecting both statistical data as well as the capture of microscopic images of the cellular localization of breast cancer related proteins.  The data collected thus far suggest that the treatment of breast cancer by various chemotherapeutics as well as ionizing radiation may in fact trigger variability in the repair process of the induced DNA double strand breaks as the timing of important breast cancer related proteins changes with treatment conditions.