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1.  Development of a High-Throughput Cell-Based Reporter Assay to Identify Stabilizers of Tumor Suppressor Pdcd4 
The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor promoting conditions. Here, we describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain (“target”) or an adjacent (“off-target”) region of Pdcd4 were cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g. TPA, p70S6K1-overactivation), while the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (> 50% inhibition of TPA-induced loss of signal, confirmed by re-assay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z’ averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers.
PMCID: PMC2853809  PMID: 19901084
Pdcd4; translation; tumor suppressor; high-throughput assay; natural products

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