Andrea J. Clark

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells Their Apparent Roles in Cell Motility and Invasion

Intracellular Ca2+ signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca2+ signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca2+-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca2+ channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell’s periphery. HT1080 cells exhibit periodic intracellular Ca2+ spikes. High-speed imaging revealed that the Ca2+ spikes were composed of a single Ca2+ wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca2+ channel blocker mibefradil inhibited Ca2+ spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd3+ and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca2+ channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca2+ wave, thereby enhancing the efficacy of emerging therapeutic protocols.

Myeloperoxidase accumulates at the neutrophil surface and enhances cell metabolism and oxidant release during pregnancy

Pregnancy is a unique immunological state. Pregnancy neutrophils differ from those of non‐pregnant women as they cannot be fully activated for oxidant production, but yet have higher levels of unstimulated oxidant production. Although reduced activation is due to decreased hexose monophosphate shunt activity, the mechanism enhancing basal oxidant levels is unknown. We hypothesize that myeloperoxidase (MPO) trafficking affects the basal oxidant release by maternal neutrophils. Immunofluorescence microscopy has demonstrated MPO at the surface of pregnancy neutrophils, whereas non‐pregnancy cells do not exhibit surface MPO. Adherent pregnancy neutrophils were characterized by high‐amplitude metabolic oscillations, which were blocked by MPO inactivation. Conversely, metabolic oscillatory amplitudes of control neutrophils were heightened by incubation with PMA or exogenous MPO. Importantly, MPO decoration of cell surfaces and high‐amplitude metabolic oscillations were observed for neutrophils from pregnant but not from non‐pregnant mice. However, cells from pregnant MPO knockout mice did not exhibit MPO expression or high‐amplitude metabolic oscillations. Unstimulated neutrophils from pregnant women were found to release reactive oxygen metabolites (ROM) and reactive nitrogen intermediates (RNI), but cells from non‐pregnant women did not. MPO inhibition returned ROM and RNI formation to non‐pregnant levels. Hence, MPO trafficking influences metabolic activity and oxidant production in pregnancy.

Calicum microdomains form within neutrophils at the neutrophil–tumor cell synapse: role in antibody-dependent target cell apoptosis

Ca2+ messages are broadly important in cellular signal transduction. In immune cells, Ca2+ signaling is an essential step in many forms of activation. Neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is one form of leukocyte activation that plays an important role in tumor cell killing in vitro and in patient care. Using fluorescence methodologies, we found that neutrophils exhibit Ca2+ signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca2+ microdomains at the neutrophil-to-tumor cell interface where they display dynamic features such as movement, fusion, and fission. These signals were blocked by the intracellular Ca2+ buffer BAPTA. At the neutrophil–tumor cell synapse, the neutrophil’s cytoplasm was enriched in STIM1, a crucial mediator of Ca2+ signaling, whereas the Ca2+-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca2+ microdomains are due to an active signaling process. As Ca2+ signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Maltooligosaccharides from JEG-3 Trophoblast-Like Cells Exhibit Immunoregulatory Properties

Citation Zhu A, Romero R, Huang J‐B, Clark A, Petty HR. Maltooligosaccharides from JEG‐3 trophoblast‐like cells exhibit immunoregulatory properties. Am J Reprod Immunol 2011; 65: 54–64

Ischemia-induced nitrotyrosine formation and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in human retinal pigment epithelium in vivo

Abstract Reactive oxidative compounds including superoxide anions and nitric oxide are believed to play a central role in many blinding eye diseases. In the present study, we examine the effect of ischemia on human retinal pigment epithelial (RPE) cells in an unusual clinical case. We show that ischemia leads to extensive nitrotyrosine deposition in the RPE and choroid, thus indicating NO-dependent oxidative stress. We also show for the first time the in vivo translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) to the nuclei of RPE cells. This enzymes nuclear translocation has previously been demonstrated in vitro where it is a marker of apoptosis. Furthermore, nitrotyrosine deposition and GAPDH translocation have been duplicated in vitro using human RPE cells. Thus, nitrotyrosine formation and GAPDH trafficking to the nucleus may be observed during ischemic conditions.

A facile method for immunofluorescence microscopy of highly autofluorescent human retinal sections using nanoparticles with large Stokes shifts.

The human retina is rich in autofluorescent species, such as lipofuscin and melanin. Consequently, it is difficult to localize antigens in the human retina using immunofluorescence microscopy. To address this issue, we have developed a methodology to tag retinal antigens using quantum dot nanoparticles that absorb in the ultraviolet and emit in the infrared, thereby avoiding the visible spectrum. This protocol dramatically improves signal-to-background autofluorescence ratios of immunofluorescence images of human retinal sections, thus enhancing the specific fluorescence in microscopic studies. Of particular note is the ability to detect antigens within the brightly autofluorescent RPE cell layer.

Differential Intracellular Distributions of Inositol Trisphosphate and Ryanodine Receptors Within and Among Hematopoietic Cells

To better understand the mechanism(s) of leukocyte Ca2+ signaling, we have studied the intracellular locations of two Ca2+-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca2+ signaling.

WO3/Pt nanoparticles are NADPH oxidase biomimetics that mimic effector cells in vitro and in vivo.

To provide a means of delivering an artificial immune effector cell-like attack on tumor cells, we report the tumoricidal ability of inorganic WO3/Pt nanoparticles that mimic a leukocytes functional abilities. These nanoparticles route electrons from organic structures and electron carriers to form hydroxyl radicals within tumor cells. During visible light exposure, WO3/Pt nanoparticles manufacture hydroxyl radicals, degrade organic compounds, use NADPH, trigger lipid peroxidation, promote lysosomal membrane disruption, promote the loss of reduced glutathione, and activate apoptosis. In a model of advanced breast cancer metastasis to the eyes anterior chamber, we show that WO3/Pt nanoparticles prolong the survival of 4T1 tumor-bearing Balb/c mice. This new generation of inorganic photosensitizers do not photobleach, and therefore should provide an important therapeutic advance in photodynamic therapy. As biomimetic nanoparticles destroy targeted cells, they may be useful in treating ocular and other forms of cancer.

Bi‐Directional Calcium Signaling Between Adjacent Leukocytes and Trophoblast‐Like Cells

Citation Elfline M, Clark A, Petty HR, Romero R. Bi‐directional calcium signaling between adjacent leukocytes and trophoblast‐like cells. Am J Reprod Immunol 2010