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DTIC ADA417511: Novel Membrane-Associated Targets for Diagnosis and Treatment of Breast Cancer

Proteins localized to the cell membrane or secreted show great promise as therapeutic targets and diagnostic markers because of their easy accessibility. However, determining protein localization by traditional methods is a difficult process. A feature of membrane-bound and secreted proteins can be exploited to determine their membrane-bound status on a large scale. Because the mRNA transcripts of membrane-bound and secreted proteins are translated in polysomes bound to the endoplasmic reticulum (ER), they can be separated from their heavier cytosolic counterparts by sucrose gradient centrifugation. At the end of year one of this project, we have reproducibly separated the RNA of MCF7 cells into two fractions using this method. Realtime RT-PCR analysis of two test genes shows enrichment of the RNA encoding cytoplasmic GAPDH in the expected fraction, and an enrichment of the RNA encoding membrane-localized JAM in the membrane fraction. Although we have experienced some difficulty in increasing RNA yield, we are confident we will soon have enough RNA to hybridize to Affymetrix chips, so that we may continue this identification on a large scale. Combined with breast cancer expression

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