Andrea L. Holme

The sulfinic acid switch in proteins

Recent studies on the redox behaviour of cysteine residues in peptides and proteins have dramatically changed our perspective of the amino acids role in biocatalysis, intracellular redox sensing and cell signalling. Cysteine sulfinic acid formation in proteins, for example, has long been viewed as an irreversible ‘overoxidation’ process that might lead to loss of activity, especially under conditions of oxidative stress. Within the last year, several research groups have independently shown that sulfinic acids can be reduced to thiols in vivo. An enzyme with sulfinic acid reductase activity, called sulfiredoxin, has been isolated from yeast and a gene encoding a human analogue has been identified in the human genome. Reversibility of sulfinic acid formation opens the door to a range of yet unexplored redox cycles, cell signalling processes and reduction mechanisms. These cysteine-based redox processes will be of enormous interest to chemists, biochemists, biologists and the medical community alike.

Towards multifunctional antioxidants: synthesis, electrochemistry, in vitro and cell culture evaluation of compounds with ligand/catalytic properties

Numerous human diseases are linked to a biochemical condition known as oxidative stress (OS). Antioxidants are therefore becoming increasingly important as potential disease prevention and therapeutic agents. Since OS is a multi-stressor event, agents combining a range of different antioxidant properties, such as redox catalysis and metal binding, might be more effective and selective than mono-functional agents. Selenium derivatives of aniline and pyridine combine redox activity with metal binding properties. These multifunctional agents have a distinct electrochemical profile, and exhibit good catalytic activity in the glutathione peroxidase mimic and metallothionein assays. They also show antioxidant activity in a skin cell model of UVA-induced stress. These compounds might therefore provide the basis for novel agents combining two or more distinct antioxidant properties.

Analysis of doppel protein toxicity

The recently described doppel protein (Dpl) is a homologue of the prion protein (PrP(c)). This protein, expressed in the brains of mice that lack the expression of PrP(c), causes neuronal death as the mice age. Previous studies have suggested this neuronal damage is caused by oxidative assault and changes in the activity of NOS proteins. We investigated the toxicity of Dpl in cell culture models and showed that Dpl was toxic to neurons. This toxicity was inhibited by the expression of PrP(c) and possibly involved direct interaction between the two proteins. The mechanism of toxicity involved stimulation of nitric oxide production via activation of the nitric oxide synthases, nNOS and iNOS. This mechanism of toxicity is quite different from that of PrP(Sc) and does not require the protein to change conformation. These results provide the first evidence for the mechanism of Dpl toxicity.

Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry

Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow‐based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated.

A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation.

The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp°/° cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP‐knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.

Automated laser scanning cytometry: A powerful tool for multi‐parameter analysis of drug‐induced apoptosis

Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi‐parameter analysis of drug‐induced tumor cell apoptosis.

Functional analyses of phosphorylation events in human Argonaute 2

Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798.

A three-dimensional co-culture system to investigate macrophage-dependent tumor cell invasion

Macrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion.

Biphasic activity of CD137 ligand-stimulated monocytes on T cell apoptosis and proliferation

CD137L (4‐1BBL) is a member of the TNFSF and is expressed on APCs as a transmembrane protein. Reverse signaling by CD137L in monocytes causes cell activation and differentiation to mature inflammatory DCs that can stimulate T cell proliferation. However, CD137L agonists have also been reported to induce apoptosis in PBMCs. This study aimed at clarifying these seemingly opposing activities. We find that the dying cells within PBMCs are T cells and that this T cell death is dependent on monocytes and correlates with the monocyte:T cell ratio. This CD137L‐induced, monocyte‐mediated T cell apoptosis is reminiscent of MDCD, and both are cell contact‐dependent. T cell death is not mediated by CD95 or DR4 or ‐5 but by ROS produced by the T cells. T cell apoptosis is restricted to the first 24 h of stimulation, and at later time‐points, the monocytes differentiate to inflammatory DCs under the influence of CD137L signaling and acquire the capacity to stimulate T cell proliferation from Day 4 onward. This biphasic activity may contribute to infection‐induced T cell attrition, where in the early phase (<24 h) of an infection, massive T cell apoptosis occurs before the antigen‐specific T cells expand.

Abstract LB-139: Laser scanning cytometry for the imaging and quantitation of membrane lipid rafts in cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The role of lipid trafficking in the physiological and patho-physiological processes is a subject of intense investigation. The enrichment of a large array of signal transduction components in caveolae and lipid rafts provide testimony to their role as signal portals. Similarly, the formation of intracellular lipid droplets, which were originally thought to be lipid storage organelles, now are known to play an important role in signaling and lipid trafficking. While these cellular components can be assessed using TPLC, mass spectrometry or western blotting, they can also be identified based on the staining of specific lip ids and proteins and analyzed by flow cytometry or using a microscope. We have employed fluorescent based techniques with the laser scanning cytometry (LSC), which allows quantification and localization of fluorescence signals as well as generating digital based images, to investigate lipid rafts and lipid droplets in 3 different cell culture models: 1) B cell lymphomas(Raji and DG75 cell lines) in which we previously reported mislocalization of the apoptosis adaptor protein APAF-1 in the lipid rafts. Nystatin, methyl-beta cyclodextrin and cytocholasin B were used to validate the labeling technique, 2) macrolipophagy, induced during autophagy, in the lung cancer cell line A549 and the cervical cancer cell line HeLa (ECACC) to evaluate the changes in lipid droplets accompanying the induction of autophagic vesicles, and 3) the formation of lipid droplets and raft structures in A549 cells following exposure to a panel of anti-cancer agents. To monitor these events, a sequential multi-colored labeling strategy was employed that allowed for the labeling of the plasma membrane using an antibody to pan-cadherin or by staining with Alexa Fluor 647 conjugated wheat germ agglutinin, prior to fixation, in combination with the labeling of lipid rafts using biotin cholera toxin B coupled with avidin conjugate Q dot 655 and antibodies to caveolin and the pro-apoptotic protein APAF-1. This labeling protocol was used in both cell cultures and in tissues. To detect the formation of lipid droplets, nile red and oil red were used to characterize the lipid content and where possible the number and localization of droplets. A threshold based segmentation strategy was employed that allows for the detection of changes to the total amount of lipid rafts, droplets, or vesicles, together with the digital maxpixel which permits monitoring of clustering or diffusion of events, and the correlation with the cell cycle. Using the profile and segmentation tools, the distance from the plasma membrane or nucleus was calculated and the localization of the events in the cytoplasm, nucleus and plasma membrane were determined using peripheral contouring and masking filters. Furthermore the digital generated images were processed using a set of image filters to generate a “3D” image of the cell or tissue giving a better resolution of events. Results provide a novel imaging strategy for the detection of lipid raft-like structures in cells and tissues, and given their importance in diverse biological systems and pathways could have potential clinical implications Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-139.