This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.IL-8 ͉ genomics ͉ high-throughput screening ͉ transducer of regulated cAMP response element-binding protein

Gene expression signatures have the ability to serve in both prognostic and predictive capacities in patient management. The use of RNA as the starting material and the lability of this analyte, however, dictate that tissues must be snap-frozen or stored in a solution that can maintain the integrity of the RNA. We compared pairs of snap-frozen and RNAlater preservativesuspended tissue from 30 such paired lymph nodenegative breast tumors and 21 such paired Dukes' B colon tumors. We assessed the correlation of gene expression profiles and prediction of recurrence based on two prognostic algorithms. Tissues stored in RNAlater preservative generated expression profiles with excellent correlation (average Pearson correlation coefficients of 0.97 and 0.94 for the breast and colon tumor pairs, respectively) compared to those produced by tissues that were snap-frozen. The correlation in the prediction of recurrence was 97% and 95% for the breast and colon tumor pairs, respectively, between these two types of tissue handling protocols. This novel finding demonstrates that prognostic signatures can be obtained from RNAlater preservative-suspended tissues, an important step in bringing gene expression signatures to the clinic. (J

BACKGROUND: Several studies have demonstrated the value of DNA methylation in urine-based assays for prostate cancer diagnosis. However, a multicenter validation with a clinical prototype has not been published.

A fluorescent imaging plate reader (FLIPR) membrane potential (V m ) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT 2 ) in a stable rGlyT 2 -HEK cell line. Data show that glycine activation of rGlyT 2 consistently results in a concentration-dependent V m response on the FLIPR that is blocked by the potent and selective GlyT 2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylaminocyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [ 3 H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT 2 physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT 2 inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96-and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V m assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT 2 . (Journal of Biomolecular Screening 2005:365-373)