Brian L. Webb

v-Src Generates a p53-Independent Apoptotic Signal

ABSTRACT Evasion of apoptosis appears to be a necessary event in tumor progression. Some oncogenes, such as c-myc and E1A, induce apoptosis in the absence of survival factors. However, others, such asbcl-2 and v-src, activate antiapoptotic pathways. For v-Src, these antiapoptotic pathways are dependent on the function of Ras, phosphatidylinositol (PI) 3-kinase, and Stat3. Here we asked whether v-Src can activate a proapoptotic signal when survival signaling is inhibited. We show that when the functions of Ras and PI 3-kinase are inhibited, v-src-transformed Rat-2 fibroblasts undergo apoptosis, evidenced by loss of adherence, nuclear fragmentation, and chromosomal DNA degradation. The apoptotic response is dependent on activation of caspase 3. Under similar conditions nontransformed Rat-2 cells undergo considerably lower levels of apoptosis. Apoptosis induced by v-Src is accompanied by a loss of mitochondrial membrane potential and release of cytochromec and is blocked by overexpression of bcl-2, indicating that it is mediated by the mitochondrial pathway. However apoptosis induced by v-Src is not accompanied by an increase in the level of p53 and is not dependent on p53 function. Thus v-Src generates a p53-independent proapoptotic signal.

A Reporter System for Reverse Transfection Cell Arrays

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conven tional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection tech nology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.

G-protein-coupled receptor microarrays for multiplexed compound screening.

Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized “plug-and-play” format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.

Fabrication and Application of G Protein-Coupled Receptor Microarrays

The increased number of drug targets and compounds demands novel high-throughput screening technologies that could be used for parallel analysis of many genes and proteins. Protein microarrays are evolving promising technologies for the parallel analysis of many proteins with respect to their abundance, location, modifications, and interactions with other biological and chemical molecules. This chapter specifically describes the fabrication of G protein-coupled receptor (GPCR) microarrays, a unique subset of protein microarrays, using contact-pin printing technology. The bioassays and potential applications of GPCR microarrays for the determination of compound affinities and potencies are also included.

Novel Surface Technologies for Genomics, Proteomics, and Drug Discovery

Following the recent progress in functional genomics and proteomics, and high-throughput screening (HTS) in drug discovery, evolving technologies over the last decade have offered a tremendous leap over the caveats of traditional techniques. In response to this metamorphosis of technologies through different platforms, Corning has introduced a suite of surface technologies with applications in microarray printing, enhanced attachment, and consumables in drug discovery. Microarrays generated on an ultra-flat glass substrate with GAPS coating exhibiting a robust chemistry and low surface background have led to higher sensitivity and reproducibility for the expression assay. Recent introduction of UltraGAPS™ surface enables oligo attachment for use in differential gene expression analysis. Various attachment surfaces to meet the needs of the applications in genomics, proteomics and drug discovery will be discussed.