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The companies in this article were selected at random. Their inclusion in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. Cell signaling, otherwise known as signal transduction, is the process by which an external stimulus (or signal) elicits a response by triggering a series of molecular events within a cell. “It is,” explains David Ferrick, vice president of biology and clinical applications at Guava Technologies, “the way the cell manages and responds to stimuli.”Recent research on the process has laid the foundation for fundamental understanding of cellular biology and has begun to influence choices made in drug discovery. “Within the past few years, scientific results achieved in cell signaling research have contributed significantly to a broader and more detailed understanding of fundamental principles such as cellular development, apoptosis, differentiation, and communication,” says Alexander Goetz, head of the tissue culture group at Greiner Bio-One. “Cell signaling is hugely critical because understanding life is about how cells communicate with one another,” adds Keith Watling, lead for the cell signaling initiative at Sigma-Aldrich. “It is at the crux of all life science research.” Jose Remacle, director of R&D and head of Eppendorf Array Technologies, and professor at the University of Namur, agrees. “For about 10 years now it has been the main issue in cell biology,” he says. “We realized that the cell’s response to the environment was important through cell signaling and changing gene expression.” The clinical community has come to the same conclusion. “People realize how important it is to deconvolute signaling pathways,” says John Anson, head of lead discovery in global development at GE Healthcare. “They realize that diseases and the treatment of them can be implemented at various levels of signaling pathways. It’s giving us more of a 3-D view of biology.” That leads inevitably to applications in drug discovery. “It’s essential in drug discovery to understand pathways from the standpoint of being cost effective,” Richard Krzyzek, head of the molecular biology group at R&D Systems, explains. “You have to narrow things down.” Jamie Baumgartner, director of assay development and global technical director for MDS Pharma Services points out the cost benefit of research on cell signaling. “For drug discovery, the cell based signaling efforts help to make more informed decisions about which drug candidates to push forward,” he says. “It’s critical to have the right cell based models.”
RAPID ADVANCES Emerging technology has made key contributions to the new understanding. “Mass spectrometry. MRI. Gels. Protein arrays. All these tools that allow us to manage complex biology have played out in cell signaling,” Ferrick says. Baumgartner adds to the list. “Higher resolution technologies such as whole cell imaging and gene display chips are really crucial to understanding what’s going on,” he says. Anson takes up the theme of improved imaging. “Very high quality microscopes like our IN Cell analyzers that can perform automated image analysis are very important,” he says. “They are moving cell biology away from being a visual science to a quantitative one.” Specialists in cell signaling have also learned to take advantage of new research techniques as soon as they become available. “RNA interference has so many advantages over knockin and knockout mice. It enables you to modulate the amount of knockdown,” Krzyzek says. “Phosphorus specific antibodies have become immense tools. And single cell methods are starting to gain a lot of popularity.” Clinical applications of cell signaling have also benefited from the use of new tools. “Better reagents and better cell based models of disease states are very important,” Baumgartner says. Goetz points to the growing understanding of intracellular signaling cascades in the induction of apoptosis and cell cycle arrest. “This understanding,” he says, “opens the door to formerly unknown and unexpected therapeutic options for the treatment of not only cancer but also autoimmune diseases, neurological dysfunctions, and many more fields of applied cell and tissue based research.” At the most fundamental level, current understanding of cell signaling owes much to the human genome project. “As a result of the project, we now know the cast of characters,” Watling explains. “It’s now a matter of figuring out which ones contribute to physiology and diseases and which we should target to find new cures and treatments.”
THE START OF SIGNALING Scientists call the extracellular signal molecule that binds to the membrane receptor the pathway’s first messenger. A second messenger is a nonprotein molecule that participates in the intracellular transduction of a signal. Cyclic AMP provides a typical example of a second messenger that mediates the actions of hormones involved in all aspects of cellular metabolism, growth, and differentiation. Its importance as a second messenger stems from the fact that it helps to regulate a large number of metabolic processes inside the cell.
Cells also use signaling mechanisms to communicate with other cells nearby, and even those in relatively remote locations in an organism. The release of hormones into the bloodstream allows cells to transmit signals to and from each other over significant distances. The initial interaction between a signal molecule and a receptor protein causes the protein to change its shape and activity. The same signal molecule may evoke different responses in different types of cells for two reasons. Cells may have different kinds of receptors or they may have different interior signal transduction pathways that connect to the same receptor. Scientists recognize several different types of receptors for external molecules. They include receptors that penetrate the plasma membrane and have intrinsic enzymatic activity, such as insulin and endothelial growth factor receptors; receptors coupled inside the cell to GTP-binding and hydrolyzing proteins, known as G proteins; and receptors that are actually ion channels, which control the flow of specific ions into the cell.
THE KINASE CONNECTION A mammalian cell might have more than 100 different kinases, mostly of the serine-threonine type that phosphorylate proteins on their serine or threonine residues. Prominent examples of serine-threonine kinases include activin and TGF-Beta receptors. A.G. Scientific, Biomol, R&D Systems, and Sigma-Aldrich offer kinase enzymes and related products. A key development in the study of kinases involved the production of specific targeted antibodies that identify the active form of protein kinases. These tools allow researchers to improve their understanding of kinases’ roles in signal transduction pathways. BD Biosciences, EMD Biosciences, R&D Systems, and Zymed Laboratories, among other companies, provide antibodies that researchers can use to study the impact of kinases on signal transduction. GPCRs form the largest family of receptors, with a membership of over 1,000. G protein mediated pathways involve at least three components: a GPCR; a G protein; and the protein that the G protein activates. Some of these receptors modulate adenylyl cyclase activity (including beta-adrenergic and glucagon receptors). G protein-coupled receptors participate in the synthesis of cAMP in response to hormonal stimulation. Researchers can obtain biochemicals and reagents for studying GPCRs from BioSource International, Sigma-Aldrich, and STI-Signal Transduction Products. Membrane receptors also include ligand-gated ion channels. These exist as protein pores in a cell’s plasma membrane that open and close in response to the binding of a chemical molecule (or ligand). This event can change the flow into the cell of such specific ions as Ca2+ and Na+. The change in the concentration of a particular ion inside the cell can often affect cellular function.
PRODUCTS AND KITS Alexis Biochemicals, Invitrogen, and Sigma-Aldrich offer a wide range of kits and reagents for biochemical assays and cell signaling studies. The products combine all the materials necessary to study a particular cellular function and eliminate some of the unknowns involved in sourcing different reagents and biochemicals from various providers. Most of the kits are use tested to ensure that researchers can learn quickly how to conduct the experiments. “We try to position ourselves as the leader in the cell signaling business,” Sigma-Aldrich’s Watling says. “We’re trying to provide all the necessary tools on a continuing basis, including small organic molecules, peptides, antibodies, recombinant proteins, and assay kits. We introduce about 600 new products a year, a large percentage of which we make ourselves.” One group of those products has specific application to the analysis of cell signaling pathways. “Our R&D team in Israel recently developed one of the first antibody arrays, called the Panorama Antibody Microarray Cell Signaling Kit,” Watling says. “It’s a collection of 224 of our antibodies that span the whole spectrum of cell signaling – apoptosis, cell cycles, cell stress, and neurobiology – and allows researchers to examine the differential expression of key cell signaling proteins in cells and tissues.” Most traditional assays require a purified cell extract, which can take several hours to prepare and needs great care to avoid altering the living cell’s contents. In addition to degrading or changing molecules with the mechanical forces that might break open a cell, the process might cause enzymatic degradation of proteins and nucleic acids via native DNase, RNase, and protease molecules. “The idea of smashing a living cell to pieces and measuring one kinase still exists, but being able to study living cells intact is now important,” GE Healthcare’s Anson says. “It’s like trying to understand how a car works by examining it running rather than starting from all the bits.” Such vendors as BD Biosciences, GE Healthcare, and PerkinElmer Life Sciences have designed systems that can process large numbers of living cells under relatively natural conditions to examine molecular interactions within cells. These systems expose living cells to a compound of interest to discover whether it interacts with the cells.
AUTOMATED ASSAYS The system has multiple uses. “It fundamentally targets the research lab,” Ferrick continues. “It is also useful in bioprocessing to grow therapeutic proteins. In the screening sector it allows individuals to perform better in terms of the meaning of a hit. And in clinical practice we’re on the road to our first approval from the U.S. Food and Drug Administration.” Other vendors offer a wide range of products for studying cellular function and signal transduction events. GE Healthcare has basic kits and reagents for research studies, as well as advanced systems and services for biotech and pharma organizations interested in streamlining their operations and in getting results more quickly. “Assays are becoming more sophisticated and enabling scientists to use live cells that are more physiologically relevant,” Anson says. “We’re trying to extend the range of cell types on which you can do assays that mimic the behavior of a cell in a particular part of the body. We’ve recently worked on how to deliver assays into a wide range of cell types using modified viruses that we infect into the cell of interest.” The company also focuses on analyzing the results of assays inside cells. Its IN Cell automated analyzer reads cells and applies sophisticated image analysis techniques to quantify what happened in the assay. “That helps us to target the interesting outliers, such as particular genes behaving abnormally and hence indicating disease states,” Anson says.
LIQUID HANDLING AND AUTOMATION At the one end of the liquid handling spectrum, hand-held pipetters have advanced from a rubber bulb attached to a glass pipette to very sophisticated electronic instruments that use disposable tips and deliver the tiniest volumes imaginable with relative ease. Some hand-held devices can be programmed to deliver exact volumes of liquid on a repetitive basis, which can be ideal for preparing a group of tubes in a rack or wells in a microwell plate. Drummond Scientific and Rainin Instrument Company offer these devices. Eppendorf has pioneered several advances in liquid handling, including those from Eppendorf Array Technologies. “Our main focus is on chips,” Remacle says. “We have launched gene expression chips that enable you to follow pathways using gene expression. Now, we are going further, providing researchers with transcription factor chips so that you can link with gene expression and with the receptor. We will provide a series of chips so that researchers will have a full range of products that they can use on the same Eppendorf platform.” Researchers working with automated systems must also ensure that the assay platform they use is compatible with automated instrumentation. Companies such as Corning, Greiner Bio-One, and Nalge Nunc specialize in plasticware that is compatible with automated devices. “Without the corresponding plasticware that fulfills the highest chemical, optical, and cell related requirements, automated work is simply impossible and standardization cannot be achieved,” explains Greiner Bio-One’s Goetz. “We offer the broadest palette of microplate products, ranging from simple 96-well plates to highly defined biological and physical surface treated 384- and 1,536-well plates, as well as devices for cell and tissue culture, protein crystallization, and bacteriology. Due to their extremely low tolerances, our products are perfectly suited for automated pipetting, handling, and imaging.”
FROM THE LAB TO THE CLINIC R&D Systems’ phospho-specific DuoSet IC (Intracellular) ELISA Development Systems offer a rapid, specific, sensitive, and economical way to measure phosphorylated proteins. “These are do-it-yourself ELISAs,” explains Krzyzek. “They are quantitative. We have kits that can quantitate total and phosphorylated kinases and offer the researcher more flexibility in adjusting assay conditions to meet specific needs compared with a validated ELISA.” The company has also set out to develop reagents for DNA damage and repair regulated by signal transduction pathways. “We’re putting out antibodies for that area, which has been largely underserved,” Krzyzek adds. Researchers can link diseases associated with abnormal cell-signaling and gene-regulation-specific protein-protein interactions with molecular drug targets. Contract research organizations such as Covance, MDS Pharma Services, and PPD offer clients molecular and cell based assay services to help determine the suitability of drug candidates as early as possible. These assays evaluate immune function, determine interleukin and interferon activities, and can measure the influence of specific molecules on tumor cell growth. “Our cell biology group is putting in place cytokine profiling capabilities,” MDS Pharma Services’ Baumgartner says. “Previously the state of the art was looking at one gene, one disease. Now we can evaluate a number of cytokines at the same time. You want to influence several cytokines simultaneously to better treat the disease and provide a more complete assessment of off-target or undesirable effects.” Beyond that, he continues, “We’re working to fill in gaps in the kinase drug discovery pipeline by providing assays that allow clients to monitor protein activity in their natural environments. Instead of measuring cellular proliferation and cytotoxicity, we now have very good tools for measuring the inhibition of kinases inside cells. We work tightly with all clients to set up the assays and services that will match their program needs and constraints.” Scientists continue to elucidate new pathways and pathway components in cell signaling. As those advances continue, they will undoubtedly lead to improved treatments for diseases caused by faults in the process of signal transduction. Peter Gwynne (//pgwynne767{at}aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner (//gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.
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Science. ISSN 0036-8075 (print), 1095-9203 (online)
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