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A collection of readily reproducible experimental methods for studying the subversion of normal cell signaling that leads to cancer. Presented in step-by-step detail to ensure successful results, the methods include proven techniques for the investigation of apoptosis and cell death, complementary protocols for manipulating and/or monitoring oncogenic signals in cancer cells, and techniques for studying protein-protein interactions. A detailed protocol for capturing pure samples of malignant cells from frozen tissue specimens and two alternative techniques for analyzing their genomic DNA is also described.
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Methods in Molecular Biology TM VOLUME 218 Cancer Cell Signaling Methods and Protocols Edited by David M. Terrian HUMANA PRESS Antimitogenic Activity of Tumor Suppression 3 1 Functional Analysis of the Antimitogenic Activity of Tumor Suppressors Erik S. Knudsen and Steven P. Angus Abstract Loss of tumor suppressors contributes to numerous cancer types. Many, but not all, proteins encoded by tumor suppressor genes have antiproliferative activity and halt cell-cycle progression. In this chapter, we present three methods that have been utilized to monitor the antimitogenic action exerted by tumor suppressors. Tumor suppressor function can be demonstrated by colony formation assays and acquisition of the flat-cell phenotype. Because of the antiproliferative action of these agents, we also present two transient assays that monitor the effect of tumor suppressors on cell-cycle progression. One is based on BrdU incorporation (i.e., DNA replication) and the other on flow cytometry. Together, this triad of techniques is sufficient to determine the action of tumor suppressors and other antiproliferative agents. Key Words: Tumor suppressor; green fluorescent protein; bromo-deoxyuridine; retinoblas