Synergistic effect of folic acid and hypericin administration to improve the efficacy of photodynamic therapy via folate receptors.

Transport systems are developed to improve the solubility of the transported drug, increase its stability, enhance its pharmacological activity and target cancer while minimising side effects. In this work, nanoporous silica particles that can be functionalized and loaded with a large number of hydrophobic molecules are proposed. The designed system was modified with folic acid to target the folic acid receptors of cancer cells. This modification enabled a higher uptake of the drug by the cells. Hypericin was selected as a hydrophobic molecule/drug with photodynamic properties suitable for diagnosis and therapy. Fluorescence microscopy and flow cytometry were used to detect the targeting and distribution of hypericin in the cancer cells. Furthermore, the combination of folic acid and hypericin has been shown to form singlet oxygen and to have a synergistic effect in improving the efficacy of photodynamic therapy. The functionalisation of the particles proposed in this work holds great potential for the delivery of hydrophobic drugs to other types of cancer cells with increased expression of the folic acid receptor to which the particles can be attached.

Effective transport of aggregated hypericin encapsulated in SBA-15 nanoporous silica particles for photodynamic therapy of cancer cells.

Photodynamic therapy (PDT) represents an interesting modality for the elimination of damaged biomaterials and cells. This treatment takes advantage of the photosensitizing properties of molecules that are active only when irradiated with light. In the present work, a dual property of hypericin, a hydrophobic molecule with high performance in photodiagnostics and photodynamic therapy, was exploited. The non-fluorescent and photodynamically inactive form of hypericin aggregates was loaded into the nanopores of SBA-15 silica particles. The synthesized particles were characterized by infrared spectroscopy, thermogravimetry, differential thermal analysis, small-angle X-ray scattering and transmission electron microscopy. Hypericin aggregates were confirmed by absorption spectra typical of aggregated hypericin and by its short fluorescence lifetime. Release of hypericin from the particles was observed toward serum proteins, mimicking physiological conditions. Temperature- and time-dependent uptake of hypericin by cancer cells showed gradual release of hypericin from the particles and active cellular transport by endocytosis. A closer examination of SBA-15-hypericin uptake by fluorescence lifetime imaging showed that aggregated hypericin molecules, characterized by a short fluorescence lifetime (∼4 ns), were still present in the SBA-15 particles upon uptake by cells. However, monomerization of hypericin in cancer cells was observed by extending the hypericin fluorescence lifetime by ∼8 ns, preferentially in lipid compartments and the plasma membrane. This suggests a promising prognosis for delayed biological activity of the entire cargo, which was confirmed by effective PDT in vitro. In summary, this work presents an approach for safe, inactive delivery of hypericin that is activated at the target site in cells and tissues.

Doxorubicin impacts chromatin binding of HMGB1, Histone H1 and retinoic acid receptor.

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and its dose-limiting side effects, especially cardiotoxicity. Here, we studied the effects of Dox on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in live cells. At lower doses, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations achieved during chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. Combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described are likely to contribute to both the effects and side effects of Dox.

Opposing Effects of Chelidonine on Tyrosine and Serine Phosphorylation of STAT3 in Human Uveal Melanoma Cells.

STAT3 is a transcription factor that regulates various cellular processes with oncogenic potential, thereby promoting tumorigenesis when activated uncontrolled. STAT3 activation is mediated by its tyrosine phosphorylation, triggering dimerization and nuclear translocation. STAT3 also contains a serine phosphorylation site, with a postulated regulatory role in STAT3 activation and G2/M transition. Interleukin-6, a major activator of STAT3, is present in elevated concentrations in uveal melanomas, suggesting contribution of dysregulated STAT3 activation to their pathogenesis. Here, we studied the impact of chelidonine on STAT3 signaling in human uveal melanoma cells. Chelidonine, an alkaloid isolated from Chelidonium majus, disrupts microtubules, causes mitotic arrest and provokes cell death in numerous tumor cells. According to our flow cytometry and confocal microscopy data, chelidonine abrogated IL-6-induced activation and nuclear translocation, but amplified constitutive serine phosphorylation of STAT3. Both effects were restricted to a fraction of cells only, in an all-or-none fashion. A partial overlap could be observed between the affected subpopulations; however, no direct connection could be proven. This study is the first proof on a cell-by-cell basis for the opposing effects of a microtubule-targeting agent on the two types of STAT3 phosphorylation.

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti-IL-2Rα therapies.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their ß- and γc-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti-IL-2 efficiently blocked IL-2-induced proliferation of IL-2-dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2-producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2-producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100-1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 µm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Minimum degree of overlap between IL-9R and IL-2R on human T lymphoma cells: A quantitative CLSM and FRET analysis.

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γc -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors. Complementing Pearson correlation data with co-occurrence analysis of confocal microscopic images revealed that a minimum degree of IL-9R/IL-2R co-localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL-9/IL-2R system. Binding of IL-9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL-9R and IL-2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines. © 2018 International Society for Advancement of Cytometry.

Experimental therapy of doxorubicin resistant human uveal melanoma with targeted cytotoxic luteinizing hormone-releasing hormone analog (AN-152).

BACKGROUND: Cytotoxic analogs of LHRH (luteinizing hormone-releasing hormone) can be successfully used for the treatment of hormone-dependent cancers such as prostatic, ovarian, endometrial, but our knowledge about their effect on hormone-independent cancers such as human uveal melanoma (UM) is limited. Previously, we have demonstrated that 46% of UM express full-length LHRH receptors. This finding has led us to further examine the mechanism of action of LHRH receptor based targeted therapies in this malignancy. AIMS: In the present study we investigated the cellular uptake of doxorubicin (DOX) and cytotoxic LHRH analog AN-152 (AEZS-108, zoptarelin doxorubicin) on human UM cell lines (OCM3) and its DOX resistant form OCM3DOX320 by confocal laser scanning microscopy. The LHRH receptor expression was characterized by RT-PCR and immunocytochemistry. RESULTS: We were able to establish a new, stable and DOX resistant human UM cell line OCM3DOX320. Our results demonstrated the expression of splice variants and isoforms of receptor for LHRH in OCM3 UM cell line and its doxorubicin resistant form OCM3DOX320. It has been revealed by MTT assay that AN-152 inhibited cell proliferation in a dose dependent manner in OCM3DOX320 cells. Furthermore, receptor-mediated uptake of AN-152 was demonstrated using confocal laser scanning microscopy in both cell line. CONCLUSIONS: Our results suggest that the antiproliferative effect of AN-152 can be detected even if only LHRH receptor isoforms are expressed. Our study also demonstrates the LHRH receptor-mediated uptake of AN-152 in DOX resistant OCM3DOX320 cells. Our experiments provide new insights into a potential targeted therapy of UM and give further details about the accumulation of AN-152 in hormone-independent DOX-resistant cells expressing splice variants of the LHRH receptors.

Membrane Potential Distinctly Modulates Mobility and Signaling of IL-2 and IL-15 Receptors in T Cells.

The high electric field across the plasma membrane might influence the conformation and behavior of transmembrane proteins that have uneven charge distributions in or near their transmembrane regions. Membrane depolarization of T cells occurs in the tumor microenvironment and in inflamed tissues because of K+ release from necrotic cells and hypoxia affecting the expression of K+ channels. However, little attention has been given to the effect of membrane potential (MP) changes on membrane receptor function. Therefore, we studied the influence of membrane de- and hyperpolarization on the biophysical properties and signaling of interleukin-2 (IL-2) and interleukin-15 (IL-15) receptors, which play important roles in T cell function. We investigated the mobility, clustering, and signaling of these receptors and major histocompatibility complex (MHC) I/II glycoproteins forming coclusters in lipid rafts of T cells. Depolarization by high K+ buffer or K+ channel blockers resulted in a decrease in the mobility of IL-2Rα and MHC glycoproteins, as shown by fluorescence correlation spectroscopy, whereas hyperpolarization by the K+ ionophore valinomycin increased their mobility. Contrary to this, the mobility of IL-15Rα decreased upon both de- and hyperpolarization. These changes in protein mobility are not due to an alteration of membrane fluidity, as evidenced by fluorescence anisotropy measurements. Förster resonance energy transfer measurements showed that most homo- or heteroassociations of IL-2R, IL-15R, and MHC I did not change considerably, either. MP changes modulated signaling by the two cytokines in distinct ways: depolarization caused a significant increase in the IL-2-induced phosphorylation of signal transducer and activator of transcription 5, whereas hyperpolarization evoked a decrease only in the IL-15-induced signal. Our data imply that the MP may be an important modulator of interleukin receptor signaling and dynamics. Enhanced IL-2 signaling in depolarized Treg cells highly expressing IL-2R may contribute to suppression of antitumor immune surveillance.

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice.

During cold-exposure 'beige' adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2+/+ and TG2-/- mice had the same amount of WAT and BAT, we found that TG2+/+ animals could tolerate acute cold exposure for 4h, whereas TG2-/- mice only for 3h. Both TG2-/- and TG2+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3h treatment; however, TG2-/- mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of 'beige' marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2-/- mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2-/- mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.

Biophysical characterization of histone H3.3 K27M point mutation.

Lysine 27 to methionine (K27 M) mutation of the histone variant H3.3 drives the formation of an aggressive glioblastoma multiforme tumor in infants. Here we analyzed how the methionine substitution alters the stability of H3.3 nucleosomes in vitro and modifies its kinetic properties in live cells. We also determined whether the presence of mutant nucleosomes perturbed the mobility of the PRC2 subunit Ezh2 (enhancer-of-zeste homolog 2). We found that K27 M nucleosomes maintained the wild-type molecular architecture both at the level of bulk histones and single nucleosomes and followed similar diffusion kinetics to wild-type histones in live cells. Nevertheless, we observed a remarkable differential recovery of Ezh2 in response to transcriptional stress that was accompanied by a faster diffusion rate of the mobile fraction of Ezh2 and a significantly increased immobile fraction, suggesting tighter chromatin binding of Ezh2 upon transcription inhibition. The differential recovery of Ezh2 was dependent on transcription, however, it was independent from K27 M mutation status. These biophysical characteristics shed more light on the mechanism of histone H3.3 K27M in glioma genesis in relation to the kinetic properties of Ezh2.

Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

Alpha-melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1ß. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1ß cytokines.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 µM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

Distinct spatial relationship of the interleukin-9 receptor with interleukin-2 receptor and major histocompatibility complex glycoproteins in human T lymphoma cells.

The interleukin-9 receptor (IL-9R) consists of an α subunit and a γ(c) chain that are shared with other cytokine receptors, including interleukin-2 receptor (IL-2R), an important regulator of T cells. We previously showed that IL-2R is expressed in common clusters with major histocompatibility complex (MHC) glycoproteins in lipid rafts of human T lymphoma cells, which raised the question about what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopy colocalization and fluorescence resonance energy transfer experiments capable of detecting membrane protein organization at different size scales revealed nonrandom association of IL-9R with IL-2R/MHC clusters at the surface of human T lymphoma cells. Accommodation of IL-9Rα in membrane areas segregated from the IL-2R/MHC domains was also detected. The bipartite nature of IL-9R distribution was mirrored by signal transducer and activator of transcription (STAT) activation results. Our data indicate that co-compartmentalization with MHC glycoproteins is a general property of γ(c) receptors. Distribution of receptor chains between different membrane domains may regulate their function.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

Tumor-specific localization of self-assembled nanoparticle PET/MR modalities.

AIM: The aim of this work was to synthesize and study in vitro and in vivo nanocarriers used as magnetic resonance imaging (MRI) contrast agents that accumulate in tumor cells specifically overexpressing folate receptors. MATERIALS AND METHODS: Nanoparticles were prepared by self-assembly of poly-γ-glutamic acid and chitosan biopolymers and were complexed with gadolinium ions. Folic acid served as a targeting molecule. Rat hepatocellular carcinoma (HeDe) cells overexpressing folate receptors were used as a model system. For in vivo experiments, HeDe cells were transplanted under the renal capsule of F344 rats. RESULTS: In vitro results showed the significant internalization of nanoparticles into HeDe cells. MRI measurements revealed that targeting nanocarriers accumulated in tumors. The MRI/PET fusion images resulted in the exact localization of tumors. CONCLUSION: The nanocarrier provides a suitable means for the early diagnosis of tumors based on their overexpression of folate receptors.

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels.

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2(-/-) mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2(-/-) mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry.

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.

Cancer cell targeting and imaging with biopolymer-based nanodevices.

We report here the synthesis, in vitro and in vivo investigation of magnetic resonance imaging (MRI) active nanoparticles, which target folate receptor overexpressing tumor cells. Self-assembled nanoparticles with a hydrodynamic size of 50-200 nm were prepared from poly-γ-glutamic acid and chitosan biopolymers with Gd-ions. The nanoparticles are biocompatible, non-toxic and stable for several months in aqueous media. In vitro assays using confocal microscopy, flow cytometry and MR imaging on HeLa human cervix carcinoma tumor cells showed that folic acid targeted nanoparticles were internalized specifically in a folate receptor dependent manner. In vivo study confirmed, that, considerable accumulation of nanosystems was found compared with the control animal represented by the MR images. Relaxometry measurements demonstrated that the nanoparticle-Gd complexes drastically change the signal intensity of the tumor cells. Because of the contrast enhancement, they are attractive candidates as potential contrast agents for a variety of diagnostic applications including early diagnosis of tumors.