January 1, 2010 (Vol. 30, No. 1)

New Assays and Advanced Imaging Methods Allow More In-Depth and Meaningful Analyses

Cell-signaling research encompasses a range of cellular mechanisms and activities, including how cells communicate with each other, how they respond to external stimuli—activators, inhibitors, or foreign entities—and how signals are transferred and received within an individual cell, including the translocation of chemical messengers across cell membranes, from cytoplasm to nucleus, and between organelles.

The ability to dissect the signaling pathways that mediate these activities will contribute to a better understanding of normal cell development, differentiation, and death, and changes relevant to specific disease processes. This knowledge will also help guide the discovery of more targeted therapeutic agents.

New techniques for studying cell signaling were explored in a series of technology workshops at the American Society for Cell Biology (ASCB) meeting held last month in San Diego.

An underlying theme of the presentations was the trend in signal-transduction studies away from Western blot-based experiments that can identify changes in protein-expression levels in cell extracts, to more quantitative and sensitive assay systems that can quantify subtle changes in protein levels, measure increases or decreases in post-translational modifications (PTMs) such as phosphorylation or methylation, interrogate targeted cell populations and monitor signaling at the level of an individual cell, and evaluate signaling activity in selected intracellular domains.

Cell-based assays and the application of advanced imaging technologies and detection reagents to cell-signaling research enable direct visualization of pathway stimulation, translocation of chemical messengers, and changes in cell morphology, cell-cycle regulation, and viability.

Furthermore, new reagents and detection methods are increasing the sensitivity of cell-signaling assays to the point where researchers no longer have to rely on artificial systems composed of engineered cell lines that overexpress selected cell-surface receptors and can begin to study signaling pathways in more physiologically relevant cell types such as primary cells and stem cells, as well as in tissue samples, model organisms, and small-animal disease models.

Scientists from PerkinElmer presented strategies to increase the sensitivity of cell-signaling assays to enable researchers to work with more physiological cell models. They highlighted the capability to detect and measure intracellular kinases and to elucidate kinase pathways using cell-based assays. These homogeneous assays can replace standard ELISAs, Western blots, and biochemical assays, and can detect endogenous full-length proteins in cell lysates in a range of cell lines including primary cells, reported the company.

Marina Bielefeld-Sevigny, Ph.D., vp and general manager, drug discovery and research reagents solutions, described how combining multiple kinase targets in pathway kits allows researchers to “determine the phosphorylation of different proteins in the kinase cascade in one experiment.”

She introduced the most recent additions to the AlphaScreen® SureFire® and AlphaLISA® platforms, based on the bead-based Alpha technology: 46 new cell-signaling pathway and biomarker research assay kits, including 24 No Wash cellular kinase and signaling-pathway kits, the latter targeting the MAPK pathway and Akt and NF-κB signaling. Compared to conventional ELISAs they offer increased sensitivity and are more readily automated, according to Dr. Bielefeld-Sevigny, who added that they also offer more quantitative results, are easier to use, and deliver faster results than Western blots.

For immunohistochemical techniques designed to detect specific proteins in tissue sections, sensitivity and low-level signal detection may be limiting factors. Proteins expressed at low levels or only by a subset of cells in a tissue sample may not generate a measurable signal using standard immunohistochemical protocols.

One approach to overcoming this problem is to greatly overexpress the target protein, generating an artificial model system. Dr. Bielefeld-Sevigny described another approach that takes advantage of recent enhancements to the company’s Tyramide Signal Amplification (TSA)™ system, which can amplify a fluorescent or chromogenic signal. TSA binds to the antibody-horseradish peroxidase (HRP) complex, initiating an amplification cycle that increases the signal produced. The technology achieves increased signal detection sensitivity while using as much as 1,000-fold less primary antibody, she reported.

In the area of imaging technology for cell-based signal-transduction assays, she emphasized that data acquisition, handling, and analysis remain the main bottlenecks, with the volume and complexity of the data making interpretation of experimental results difficult and time consuming.

“The challenge for cellular imaging today is acquiring images with the right sensitivity, resolution, and speed. Image analysis, particularly for 3-D and live-cell imaging, become a large-scale computing problem, often requiring a 64-bit computing system solution.”

Image-analysis software is evolving to increase the speed of image acquisition and presentation, automate qualitative and quantitative analytical techniques, and simplify user interaction with the software’s data-management and analytical tools so researchers can more easily customize image visualization and analysis. Dr. Bielefeld-Sevigny reported that recent improvements to PerkinElmer’s Volocity® analysis software, working with the VoX 3D live-cell imaging system for example, allow for 3-D and 4-D image capture and visualization.

In an effort to facilitate sharing and comparison of images among researchers within or between academic laboratories, industry, and government research centers, PerkinElmer is participating in a consortium working to develop standardized formats for importing images. These standards are incorporated in the company’s Columbus™ software package for data management, storage, and retrieval.

Cell-based assays and the application of advanced imaging technologies and detection reagents to cell-signaling research enable direct visualization of pathway stimulation. (KaYann/Fotolia)

Detecting Phosphorylation Events

Debra MacIvor, Ph.D., product manager for the EpiQuant product line at Millipore, identified one of the biggest challenges in cell-signaling research as the need for more quantitative analysis of large numbers of site-specific phosphoproteins in parallel and in a range of cell phenotypes to enable the detection of multiple events in a single signaling cascade or across signal-transduction pathways. She emphasized the value of multiplexing to capture and discriminate between as many different protein targets as possible in one experiment.

Dr. MacIvor highlighted multiplex capability and absolute quantification in the picomolar range as key features of the  EpiQuant cell-signaling portfolio, which combines EpiQuant assay technology, part of the Milliplex® MAP (Multi-Analyte Profiling) platform, with Luminex detection technology.

This quantitative, multiplexed immunoassay technique can be used to obtain measurements of phosphorylation concentrations at multiple sites on a particular protein target, and to compare the total amount of target protein with the amount of phosphorylated protein, Dr. MacIvor noted. With absolute quantification it is also possible to compare changes in protein phosphorylation among different cell types in the same experiment, she added.

High multiplex capability and absolute quantification are achieved by detecting short peptides resulting from the well-characterized digestion of a cell lysate. Protein fragmentation is done with cuts made at unique linear sequences to yield a predictable set of peptides with known phosphorylation sites. EpiQuant incorporates antibodies that recognize those sequences to capture specific phosphoproteins. These same peptide sequences are the basis for generating peptide standards that serve as internal controls.

“These standards are a significant departure from the existing Luminex signaling assays and they enable quantification,” said David Hayes, director of research and development at Millipore. As the EpiQuant technology relies on sequence specificity to capture peptides, it can distinguish between human and mouse proteins and could theoretically be used in xenograft studies to evaluate signaling pathways in human tissues grown in mice.

The initial EpiQuant kit targets phosphotyrosine signaling and includes a panel of 40 phosphotyrosine analytes. Millipore plans to introduce additional EpiQuant cell-signaling panels and these will include analytes for detecting additional phosphotyrosines, receptor tyrosine kinases, phosphoserines, and phosphothreonines, as well as the capability to analyze other types of PTMs, including methylation and acetylation.

Quantification of intracellular signaling proteins in cell-based assays using PTM-specific antibodies was the focus of a workshop presented by Thermo Fisher Scientific. Brian Webb, Ph.D., platform manager, described applications for profiling signaling-protein activation with fluorescent or colorimetric readouts using the company’s in-cell ELISA technique, which he referred to as “a Western blot in a well,” but with the ability to yield quantitative data and to be automated for high-throughput applications. He noted that the in-cell ELISA also requires much fewer cells than a Western blot, preserving resources.

“With the new trend in the market in which researchers are using primary cells, stem cells, and other more scarce and valuable cells, they need a tool to screen for signal transduction more cost effectively.”

Compared to a conventional sandwich ELISA, an in-cell ELISA requires only one antibody, he added. Once the cells are grown in the wells of a microtiter plate and exposed to a drug compound, they are fixed with formaldehyde, essentially locking the physiology in time. Incubation with a primary antibody allows detection of the protein or phosphorylated/modified protein of interest.

Dr. Webb presented data demonstrating use of the technology to monitor activation of proteins in the downstream signaling cascade resulting from activation of epidermal growth factor receptor (EGF-R) by EGF. In studies comparing neuroblastoma SY5Y cells treated with brain-derived neurotrophic factor to untreated cells the assays demonstrated differential activation of 10 different signaling proteins in one experiment.
The two types of in-cell ELISA kits currently available allow for the assay to be performed with either absorbance or near-infrared detection instrumentation. For a colorimetric readout, the kit includes an HRP-conjugated detection reagent, and absorption is measured at 450 nm. The disadvantage of one-color detection is that it limits the assay to measurement of only one protein per well. The kit, designed to yield fluorescent readouts, allows for detection of two targets per well using two different secondary antibodies and two fluorescent dyes, so data acquisition and normalization can take place in the same well.

Going with the Flow

The focus of a presentation by BD Biosciences was on multicolor flow-cytometric methods for characterizing signaling mechanisms that mediate innate and adaptive immunity. Specifically, the scientists described studies comparing the signaling capacities of subsets of treated leukocytes (human CD4+ and CD8+ T-cells) by measuring intracellular phosphorylation events involved in various kinase-signaling pathways.

This research illustrates the effort at BD to transition flow cytometry into signal transduction as an alternative to Western blot technology. This effort required the development of nearly 100 antibodies to specific phosphorylation sites that are amenable to a flow-cytometry platform. It also required a method to fix and permeabilize cells and an overall protocol that could be built into a kit format for ease of use. In February, the company will introduce BD Phosflow kits designed to help researchers study phosphorylation in activated T cells in flow cytometry-based experiments.

The technology provides a straightforward method for analyzing phosphorylation events in specific cell populations isolated from heterogeneous samples, such as blood, using cell surface marker-specific antibodies. Researchers can use the technology “to detect heterogeneous signaling responses within subsets of a cell population,” said Erika O’Donnell, Ph.D., R&D scientist. Data presented at the meeting demonstrated the ability to identify similarities and differences in how leukocyte subsets responsible for innate and adaptive immunity respond to a particular stimulus.

“The technology is applicable across species and across multiple cell types, including cell lines and primary cells, and it has been applied to adherent cells,” said Jurg Rohrer, R&D director of research immunology. The initial kit targets CD4+ and CD8+ T cells; subsequent products will target additional immune cell types, including natural killer cells and monocytes.

Dose-response experiment in human whole blood analyzing intracellular phosphorylation levels in T-cell subsets: Treated human blood was processed using the BD Phosflow T Cell Activation Kit.

Measuring Gene Expression

The end result of signal transduction and activation of cell-signaling pathways is typically targeted up- or downregulation of gene expression. Improved methods for measuring changes in gene-expression levels in cell-based assays have three main goals: increased sensitivity, dynamic range, and ease of use.

Three technology platforms developed at SABiosciences can reportedly be applied to achieve these goals, as described during an ASCB workshop. With customized real-time PCR Arrays, researchers can select a panel of genes relevant for a particular pathway and assess each gene in an individual well of a microtiter plate. The 84 genes available include cell-function and disease-relevant panels, as well as controls for data normalization, for detecting genomic contamination, and for assessing PCR reactivity.

The Signal Transduction PathwayFinder™ technology has been used, for example, to identify the oncogenic pathways activated in a knockout mouse model that has a tumor-like phenotype affecting various organs. The technology generates a profile of expressed genes that control cell processes through overlapping signaling pathways composed of extracellular ligands, ligand receptors, and intracellular-signaling modulators. For these experiments, a cancer-related panel of genes was evaluated to identify which pathway(s) is up- or downregulated in the knockout mouse.

Whereas the PCR Arrays reflect changes in gene expression, the company’s Cignal™ Pathway Reporter system allows researchers to assess pathway activity, explained Brian McNally, Ph.D., associate product manager. “We view them as complementary,” one can be used to identify a relevant pathway and the other to determine what genes in the pathway have altered expression. The Cignal system relies on 45 luciferase or green fluorescent protein (GFP)-based transcription factor reporters to detect and measure cell-signaling events. The system can be used to probe single pathways or up to 10 pathways in parallel in one microtiter plate.

“Instead of looking at the expression of a single gene or at phosphorylation at a particular point in a pathway, the Cignal Reporter represents a summation of the signaling events upstream,” says Dr. McNally. It can report on positive or negative regulation of pathway activity. SABiosciences offers the reporters in a lentiviral delivery system “to allow researchers to do experiments in their own biological systems,” including studies in primary cells, stem cell clusters, or animal models.

At the ASCB meeting, SABiosciences introduced its Biology-on-Array platform for identifying proteins that regulate gene expression. The system incorporates synthetic siRNA molecules that target up to 672 transcription factors to achieve gene knockdown. After siRNA treatment, total RNA from each knockdown is converted to cDNA and real-time PCR is used to measure changes in the expression of selected genes. The Biology-on-Array software analyzes the results and identifies the transcription factors, epigenetic modification enzymes, or signaling pathways that regulate the expression of specific genes.

Dr. McNally presented an application in which the Biology-on-Array technology was used to identify 33 genes that had significantly altered expression following treatment of MCF7 breast cancer cells with the chemotherapeutic agent 5-fluorouracil (5FU): 19 genes had significantly increased expression and 14 genes were repressed. Further interrogation of one of these genes, CDKN1A, which exhibited upregulation in response to 5FU, led to the isolation of three positive regulators and two less potent negative regulators. 

A CDKN1A-specific qRT-PCR assay on 88 samples showed that, under the influence of 5-FU, knockdown of TP53 significantly downregulated CDKN1A expression. (SABiosciences)

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