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.
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.