Microarrays: spotlight on gene function and pharmacogenomics

The introduction of microarray technology has dramatically changed the way that researchers address many biomedical questions. DNA microarrays can measure expression of thousands of genes simultaneously, providing extensive information on gene interaction and function. Microarray technology is a powerful tool for identifying novel molecular drug targets and for elucidating mechanisms of drug action. Furthermore, microarrays can monitor the global profile of gene expression in response to specific pharmacologic agents, providing information on drug efficacy and toxicity. Over the last several years, dramatic advancements have occurred in array technology. In this review we describe basic aspects of microarray instrumentation and experimentation. Each of the major array formats including oligonucleotides arrays, spotted arrays, and macroarrays are examined, and advantages and options for using each format are presented. Important factors in the design and analysis of microarray experiments are also discussed. Most importantly, we explore recent developments in microarray technology that are relevant to pharmacogenomics and the discovery of gene function.     

Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions

The most challenging and fruitful biomedical research endeavor of this decade will be the mapping of cell signaling systems and establishing their linkages to normal and disease-related processes. Amongst other things, the Human Genome Sequencing Project has greatly facilitated MALDI-TOF mass spectrometry identification of proteins that have been resolved by standard 2D gel electrophoresis. However, the low abundance of protein kinases and other signal transduction proteins has rendered their analyses particularly problematic without some means of purification and enrichment from cell and tissue lysates. Antibodies have been the most specific affinity probes for tracking target proteins, but their variable quality and high cost preclude their deployment in most discovery-based proteomics studies. Current multi-immunoblotting techniques can permit the probing of a single mini-SDS-PAGE gel with 50 or more antibodies at a time to monitor large changes in the expression and phosphorylation states of signaling proteins. The development of new affinity probes to replace antibodies is necessary to drive large scale proteomics studies. Such affinity probes could include short peptide antibody mimetics (PAM's) and oligonucleotide aptamers that when spotted in 2D array formats (e.g. membrane macroarrays, glass microarrays) or presented on specific beads (e.g. Luminex beads) can capture target proteins for their specific enrichment. The bound target proteins can then be detected using reporter antibodies or other specific probes for their quantitation by high throughput systems. These new proteomics methodologies will accelerate assessment of specific protein expression, post-translational modification, protein-protein interactions and protein-drug interactions to provide a more holistic view of cellular operations and how they might be manipulated under pathological circumstances.     

Protein array technology. Potential use in medical diagnostics

The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogs and functional catalogs. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.     

The coming impact of gene expression profiling on the diagnosis and treatment of HCV-associated liver disease.

Gene expression profiling allows the level of activity of thousands of genes to be monitored simultaneously. Profiling is often carried out on specialized chips or slides, which have microarrays of gene targets at predetermined addresses. In the immediate future, microarrays promise to yield new insights into hepatitis C virus (HCV) pathogenesis and to produce 'signatures' that can be used in molecular diagnostics. In the longer-term, they may aid the development of serological tests by identifying genes encoding secretory proteins produced by HCV-infected livers, and they may suggest new avenues for disease intervention by detecting genes whose products are retained in the infected liver.     

Carbohydrate microarrays as tools in HIV glycobiology

Progress in carbohydrate microarray technology has positioned the glycochip among the expanding set of biophysical tools available to researchers. Synthetically-derived glycochips unite established microarray techniques with the versatility and structural precision of synthetic carbohydrate chemistry. A comprehensive demonstration of carbohydrate microarrays is illustrated by the chip-based study of protein/carbohydrate and protein/glycoprotein interactions as they relate to HIV glycobiology. Composed of a series of high-mannose oligosaccharides, carbohydrate microarrays were prepared utilizing a covalent linking strategy to immobilize synthetically-defined glycans in a uniform orientation. In concert with a simple glycoprotein array, these microarrays were used to establish the individual and competitive binding profiles of five gp120 binding proteins--DC-SIGN, CD4, 2G12 cyanovirin-N, and scytovirin--and established the carbohydrate structural requirements for these interactions.     

DNA microarrays in neuropsychopharmacology

Recent advances in experimental genomics, coupled with the wealth of sequence information available for a variety of organisms, have the potential to transform the way pharmacological research is performed. At present, high-density DNA microarrays allow researchers to quickly and accurately quantify gene-expression changes in a massively parallel manner. Although now well established in other biomedical fields, such as cancer and genetics research, DNA microarrays have only recently begun to make significant inroads into pharmacology. To date, the major focus in this field has been on the general application of DNA microarrays to toxicology and drug discovery and design. This review summarizes the major microarray findings of relevance to neuropsychopharmacology, as a prelude to the design and analysis of future basic and clinical microarray experiments. The ability of DNA microarrays to monitor gene expression simultaneously in a large-scale format is helping to usher in a post-genomic age, where simple constructs about the role of nature versus nurture are being replaced by a functional understanding of gene expression in living organisms.     

Genomics and proteomics: the new millennium of drug discovery and development

One of the most pressing issues facing the pharmaceutical and biotechnology industry is the tremendous dropout rate of lead drug candidates. Over the last two decades, several new genomic technologies have been developed in hopes of addressing the issues of target identification and lead candidate optimization. Gene expression microarray is one of these technologies and this review describes the four main formats, which are currently available: (a) cDNA; (b) oligonucleotide; (c) electrokinetic; and (d) fiberoptic. Many of these formats have been developed with the goal of screening large numbers of genes. Recently, a high-throughput array format has been developed where a large number of samples can be assayed using arrays in parallel. In addition, focusing on gene expression may be only one avenue in preventing lead candidate failure. Proteomics or the study of protein expression may also play a role. Two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with mass spectroscopy has been the most widely accepted format to study protein expression. However, protein microarrays are now being developed and modified to a high-throughput screening format. Examples of several gene and protein expression studies as they apply to drug discovery and development are reviewed. These studies often result in large data sets. Examples of how several statistical methods (principal components analysis [PCA], clustering methods, Shannon entropy, etc.) have been applied to these data sets are also described. These newer genomic and proteomic technologies and their analysis and visualization methods have the potential to make the drug discovery and development process less costly and more efficient by aiding to select better target and lead candidates.     

Cell-based microarrays: current progress, future prospects

Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a novel method for performing high-throughput screens of gene function. In this study, expression vectors containing the open reading frame of human genes were printed onto glass microscope slides to form a microarray. Transfection reagents were added pre- or post-spotting, and cells grown over the surface of the array. They demonstrated that cells growing in the immediate vicinity of the expression vectors underwent 'reverse transfection', and that subsequent alterations in cell function could then be detected by secondary assays performed on the array. Subsequent publications have adapted the technique to a variety of applications, and have also shown that the approach works when arrays are fabricated using short interfering RNAs and compounds. The potential of this method for performing analyses of gene function and for identifying novel therapeutic agents has been clearly demonstrated, and current efforts are focused on improving and harnessing this technology for high-throughput screening applications.     

An update on using protein microarrays in drug discovery

Protein microarrays are evolving as useful tools for biopharmaceutical research. The differences in characteristics of individual proteins has made development challenging compared with DNA arrays. Nonetheless, significant advances have nontheless been made in developing protein microarray technology. Retention of function has been demonstrated for proteins belonging to various structural and functional classes after arraying. Focused arrays with small groups of proteins have been developed for a variety of applications, from biomarker validation to small molecule screening. Issues of protein stability as well as assay specificity and sensitivity, are being worked out for panels of arrayed proteins. The development of robust manufacturing methods has resulted in an increase in the number of commercially available protein array products. Quality control guidelines, which will also aid in accelerating development of the technology, are being established.     

Natural polyphenols based new therapeutic avenues for advanced biomedical applications

Polyphenols are naturally occurring, synthetic or semisynthetic organic compounds that offer a vast array of advanced biomedical applications. The mostly researched polyphenolic compounds are resveratrol and flavanols, notably (?)-epicatechin. The ongoing research on clinically important resveratrol and flavanols has revealed their potentials as extremely efficient drug agents that can be leveraged for new therapeutic designs for combating stroke related injuries, cancer and renal failures. Here, we have highlighted recent developments in this area with an emphasis on the biomedical applications of polyphenols. Also, a perspective on the future research directions has been discussed. We believe that this review would facilitate further research and development of polyphenols as a therapeutic avenue in medical science.     

Application of recombinant and non-recombinant peptides in the determination of tumor response to cancer therapy

An early and reliable assessment of therapeutic efficacy during the treatment of cancer is essential to achieve an optimal treatment regimen and patient outcome. The use of labeled peptides to monitor tumor response is associated with several advantages. For example, peptides are very stable, non-immunogenic, are easy to label for imaging, they undergo rapid clearance from the circulation, can penetrate tumor tissue, and are inexpensive to synthesize. In this review, studies using recombinant and non-recombinant peptides to monitor the response of glioblastoma multiforme, lung, breast, pancreas, colon, prostate, and skin carcinomas to radiation and/or chemotherapeutics such as camptothecin, doxorubicin, etoposide, 5-fluorouracil, paclitaxel, AG3340, sunitinib, and dasatinib, are presented. A consideration of the imaging techniques available to monitor peptide localization, including near-infrared (NIR) fluorescence, magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasonography, is also included. Peptides that have been successfully used to monitor various tumor types and therapies have been shown to target proteins that undergo changes in expression in response to treatment, endothelial cells that respond to radiation, or mediators of apoptosis. Peptides that are able to selectively bind responsive versus unresponsive tumors have also been identified. Therefore, the advantages associated with the use of peptides, combined with the capacity for selected peptides to assess tumor response as demonstrated in various studies, support the use of labeled peptides to evaluate the effectiveness of a given cancer therapy.     

Gene-expression microarrays provide new prognostic and predictive tests for breast cancer

Wide availability of systemic therapy agents has led to a considerable decline in mortality from breast cancer. However, the biology of breast cancer remains poorly understood. Currently, highly accurate markers to predict prognosis and probability of response to a given systemic therapy on an individual basis are lacking, and routinely used clinicopathologic variables fail to fully capture the heterogeneity of breast cancer. As a result, many patients are overtreated, whereas others may not receive the necessary therapy. It has been hypothesized that molecular differences in breast cancers might account for the heterogeneous potential in growth, invasion and metastasis in each individual tumor. Gene-expression microarrays have been extensively applied in breast cancer research in the hope that a combination of multiple genes (i.e., gene signatures) will more informatively predict disease outcome and response to a specific systemic therapy. This technique holds substantial promise for optimizing clinical decision making and tailoring therapeutic regimens to individual patients in the near future. This review focuses on the recent progression in feasibility and reliability of gene-expression microarrays in identifying new prognostic and predictive indicators of breast cancer.     

Global gene expression in classification, pathogenetic understanding and identification of therapeutic targets in acute myeloid leukemia

Global gene expression analysis by way of DNA microarrays and real time quantitative PCR provides an important supplement to established diagnosis and classification of malignant disease. A comprehensive molecular understanding of the regulatory modules involved in carcinogenesis should also be important for improved identification of therapeutic targets and thus for future individualized therapy, e.g., by allowing therapeutic synergy when designing combination therapy against vulnerable points in the malignant cells. The therapeutic potential of knowledge obtained from global gene expression analysis of malignant cells is crucially dependent upon a similarly fine-grained knowledge of gene regulation in normal cells. The deviant gene expression patterns should therefore be assessed on a background of gene expression associated with housekeeping functions, particular differentiation stages and epiphenomena due to genomic instability. Since malignant cells originate from transformed precursor cells, such reference information can be obtained from investigations of embryonic and somatic stem cells. Much has recently been learned about the regulatory modules of normal hematopoietic stem cells and their malignant counterparts, and new biologically and clinically relevant patient subgroups as well as novel prognostic and therapeutic molecular markers have been identified. The present review weighs up the results and their potentialities with reference to gene expression analysis in acute myeloid leukemia (AML).     

Pretty subunits all in a row: using concatenated subunit constructs to force the expression of receptors with defined subunit stoichiometry and spatial arrangement

The members of the Cys-loop ligand-gated ion channel (LGIC) gene family play a major role in fast synaptic transmission, and these receptors represent an important class of targets for therapeutic agents. Each member of this gene family is a pentameric complex containing one or more different subunits, and a large number of subunits for each member have been identified. This large number of subunits could give rise to a bewildering array of possible subunit compositions and spatial arrangements within a single complex, not all of which may occur in vivo. Heterologous expression systems have been used to create specific combinations of individual subunits to mimic naturally occurring receptors. However, this approach is not without its problems. In this issue of Molecular Pharmacology, Groot-Kormelink et al. (page 559) describe a method for constructing "concatameric" receptors, in which five individual subunits are arranged in a predetermined order connected by a flexible linker. Expression of this construct results in the formation of receptors with a unique, predefined subunit stoichiometry and subunit arrangement within the receptor complex. Receptors formed from this construct are fully functional and have properties essentially identical to those formed from individual subunits. The application of this very general approach to other members of the LGIC family should markedly enhance our ability to understand how subunit composition influences receptor function, as well as provide a means for the expression of receptors of predefined subunit composition and arrangement as tools for the development of novel selective pharmacological and therapeutic agents.