Marie A. Iannone

Multiplexed microsphere-based flow cytometric assays.

Flow cytometry has become an indispensable tool for clinical diagnostics and basic research. Although primarily designed for cellular analysis, flow cytometers can detect any particles in the lower micron range, including inert microspheres of different sizes, dyed with various fluorochromes. Over the past 20 years, microspheres have been used as calibrators for flow cytometers and also as a solid support for numerous molecular reactions quantitated by flow cytometry. Proteins, oligonucleotides, polysaccharides, lipids, or small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule. More recently, assays for similar analytes have been multiplexed, or analyzed in the same assay volume, by performing each reaction on a set of microspheres that are dyed to different fluorescent intensities and, therefore, are spectrally distinct. Some recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interactions of nuclear receptors. The speed, sensitivity, and accuracy of flow cytometric detection of multiple binding events measured in the same small volume have the potential to replace many clinical diagnostic and research methods and deliver data on hundreds of analytes simultaneously.

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres.

BACKGROUND We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production.

Discovery of novel nuclear receptor modulating ligands: an integral role for peptide interaction profiling

There is currently a marketed drug for nearly every nuclear receptor for which the natural ligand has been identified. However, because of the complexity of signal transduction by this class of ligand-regulated transcription factors, few of these drugs have been optimized for pharmaceutical effectiveness. Over the past several years, structural and biochemical work has shed light on some of the ligand-induced features of nuclear receptors that enable them to trigger signal transduction cascades. This review will highlight the use of peptide interactions to cluster different classes of ligands and to identify novel nuclear receptor-modulating ligands as potential drug candidates. Phage display and a multiplexed peptide interaction assay are two of the technologies that are key to this approach. When used as part of a drug discovery platform, this type of biochemical characterization can bridge the gap between high-throughput chemical synthesis and disease model testing. Furthermore, the development of these methodologies is timely because there is a significant medical need for new and improved nuclear receptor drugs that retain beneficial effects but do not have undesired side effect activities.

Effect of microsphere binding site density on the apparent affinity of an interaction partner

Flow cytometric microsphere‐based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner.

Studies Concerning the Mechanism of Action of 3-Deazaadenosine in Leukocytes

3-Deazaadenosine (c3Ado) is a fascinating compound that exhibits interesting biological activities but whose mechanism of action remains an enigma. Although the synthesis of c3Ado was first reported in 1966 (Rousseau et al., 1966), during the subsequent eleven years little was reported concerning biological activities of this adenosine analogue. During this interval, c3Ado was shown to be resistant to deamination by adenosine deaminase (Ikehara & Fukui, 1974) and to be nontoxic to tumor cells (Kitano et al., 1975; May & Townsend, 1975). In 1977, however, widespread interest in c3Ado was awakened by the discovery that c3Ado is both a substrate and a potent inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (Chiang et al., 1977); in this same report it was shown that c3Ado caused a buildup of AdoHcy and was metabolized to S-3-deazaadenosylhomocysteine (c3AdoHcy) in rat hepatocytes. Following this landmark publication, numerous reports began to appear describing diverse biological effects of c3Ado, particularly with respect to leukocyte functions (reviewed by Ueland, 1982, and by Chiang, 1985). In many of these experimental Systems, exogenously supplied L-homocysteine (Hcy) has been found to enhance the biological activity of c3Ado.

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.

Taxol inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced human neutrophil polarization and H2O2 production while decreasing [3H]FMLP binding

We have studied the effect of taxol on two N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil functions and the possible mechanism by which it inhibits these functions. Taxol inhibited FMLP-induced human neutrophil polarization (a characteristic change in neutrophil shape in response to a chemotactic stimulus) and H2O2 generation. Taxol also decreased the specific binding of [3H]FMLP to human neutrophils at 4°C. The decreased binding of FMLP to its receptor may be responsible for the inhibition by taxol of FMLP-induced polarization and H2O2 generation.

Effects of adenosine on neutrophil polarization induced by N-formyl-methionyl-leucyl-phenylalanine, sodium propionate and colchicine

Polarization of human neutrophils (a characteristic bipolar shape change) can be induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP); sodium propionate, which causes a rapid acidification of the cytosol; or colchicine, which disrupts microtubules. We have previously reported that adenosine, endogenously produced in human neutrophil suspensions, inhibits FMLP-induced polarization. We report here that endogenously produced adenosine also inhibits sodium propionate-induced polarization but has no effect on colchicine-induced polariaation. These results suggest that neutrophil polarization may be a multistep process inducible by compounds that trigger different biochemical events.

Characterization of Nuclear Receptor Ligands by Multiplexed Peptide Interactions

This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere‐based system employs peptide‐coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide–nuclear receptor interactions may be analyzed in a single well of a 96‐well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein–peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no‐wash format and the high surface density of the microsphere‐coupled interaction partner offer a moderately high‐throughput system to examine low‐ to high‐affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.