Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Taking molecular pathology to the next level: Whole slide multicolor confocal imaging with the Pannoramic Confocal digital pathology scanner.

The emergence and fast advance of digital pathology allows the acquisition, digital storage, interactive recall and analysis of morphology at the tissue level. When applying immunohistochemistry, it also affords the correlation of morphology with the expression of one or two specific molecule of interest. The rise of fluorescence pathology scanners expands the number of detected molecules based on multiplex labeling. The Pannoramic Confocal (created by 3DHistech, Hungary) is a first-of-the-kind digital pathology scanner that affords not only multiplexed fluorescent detection on top of conventional transmission imaging, but also confocality. We have benchmarked this scanner in terms of stability, precision, light efficiency, linearity and sensitivity. X-Y stability and relocalisation precision were well below resolution limit (≤50 nm). Light throughput in confocal mode was 4-5 times higher than that of a point scanning confocal microscope, yielding similar calculated confocal intensities but with the potential for improving signal to noise ratio or scan speed. Response was linear with R2 ≥ 0.9996. Calibrated measurements showed that using indirect labeling ≥2000 molecules per cell could be well detected and imaged on the cell surface. Both standard-based and statistical post-acquisition flatfield corrections are implemented. We have also measured the point spread function (PSF) of the instrument. The dimensions of the PSF are somewhat larger and less symmetric than of the theoretical PSF of a conventional CLSM, however, the spatial homogeneity of these parameters allows for obtaining a specific system PSF for each optical path and using it for optional on-the-fly deconvolution. In conclusion, the Pannoramic Confocal provides sensitive, quantitative widefield and confocal detection of multiplexed fluorescence signals, with optical sectioning and 3D reconstruction, in addition to brightfield transmission imaging. High speed scanning of large samples, analysis of tissue heterogeneity, and detection of rare events open up new ways for quantitatively analyzing tissue sections, organoid cultures or large numbers of adherent cells.

The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC.

Despite recent advances in the development of novel personalized therapies, breast cancer continues to challenge physicians with resistance to various advanced therapies. The anticancer action of the anti-HER2 antibody, trastuzumab, involves antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells. Here, we report a repurposing screen of 774 clinically used compounds on NK-cell + trastuzumab-induced killing of JIMT-1 breast cancer cells. Using a calcein-based high-content screening (HCS) assay for the image-based quantitation of ADCC that we have developed and optimized for this purpose, we have found that the multitargeted tyrosine kinase inhibitor sunitinib inhibits ADCC in this model. The cytoprotective effect of sunitinib was also confirmed with two other assays (lactate dehydrogenase release, and electric cell substrate impedance sensing, ECIS). The drug suppressed NK cell activation as indicated by reduced granzyme B deposition on to the target cells and inhibition of interferon-γ production by the NK cells. Moreover, sunitinib induced downregulation of HER2 on the target cells' surface, changed the morphology and increased adherence of the target cells. Moreover, sunitinib also triggered the autophagy pathway (speckled LC3b) as an additional potential underlying mechanism of the cytoprotective effect of the drug. Sunitinib-induced ADCC resistance has been confirmed in a 3D tumor model revealing the prevention of apoptotic cell death (Annexin V staining) in JIMT-1 spheroids co-incubated with NK cells and trastuzumab. In summary, our HCS assay may be suitable for the facile identification of ADCC boosting compounds. Our data urge caution concerning potential combinations of ADCC-based immunotherapies and sunitinib.

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi.

While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe morphological changes or molecular events in fungi, especially when long observation periods or high light fluxes are warranted, encounter problems owed to altered regulatory pathways or even cell death caused by various photosensing mechanisms. Consequently, the ever expanding repertoire of visible fluorescent protein toolboxes and high-resolution microscopy methods designed to investigate fungi in vitro and in vivo need to comply with an additional requirement: to decrease the unwanted side effects of illumination. In addition to optimizing exposure, an obvious solution is red-shifted illumination, which, however, does not come without compromises. This review summarizes the interactions of fungi with light and the various molecular biology and technology approaches developed for exploring their functions on the molecular, cellular, and in vivo microscopic levels, and outlines the progress towards reducing phototoxicity through applying far-red and near-infrared light. KEY POINTS: • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity.

Anandamide Concentration-Dependently Modulates Toll-Like Receptor 3 Agonism or UVB-Induced Inflammatory Response of Human Corneal Epithelial Cells.

Photodamage-induced and viral keratitis could benefit from treatment with novel nonsteroid anti-inflammatory agents. Therefore, we determined whether human corneal epithelial cells (HCECs) express members of the endocannabinoid system (ECS), and examined how the endocannabinoid anandamide (AEA, N-arachidonoyl ethanolamine) influences the Toll-like receptor 3 (TLR3) agonism- or UVB irradiation-induced inflammatory response of these cells. Other than confirming the presence of cannabinoid receptors, we show that endocannabinoid synthesizing and catabolizing enzymes are also expressed in HCECs in vitro, as well as in the epithelial layer of the human cornea in situ, proving that they are one possible source of endocannabinoids. p(I:C) and UVB irradiation was effective in promoting the transcription and secretion of inflammatory cytokines. Surprisingly, when applied alone in 100 nM and 10 µM, AEA also resulted in increased pro-inflammatory cytokine production. Importantly, AEA further increased levels of these cytokines in the UVB model, whereas its lower concentration partially prevented the transcriptional effect of p(I:C), while not decreasing the p(I:C)-induced cytokine release. HCECs express the enzymatic machinery required to produce endocannabinoids both in vitro and in situ. Moreover, our data show that, despite earlier reports about the anti-inflammatory potential of AEA in murine cornea, its effects on the immune phenotype of human corneal epithelium may be more complex and context dependent.

A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts.

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

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.

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes.

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

Cancer-derived exosomes from HER2-positive cancer cells carry trastuzumab-emtansine into cancer cells leading to growth inhibition and caspase activation.

BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present also on cancer-derived exosomes. We hypothesized that exosome-bound T-DM1 may contribute to the activity of T-DM1. METHODS: Exosomes were isolated from the cell culture medium of HER2-positive SKBR-3 and EFM-192A breast cancer cells, HER2-positive SNU-216 gastric cancer cells, and HER2-negative MCF-7 breast cancer cells by serial centrifugations including two ultracentrifugations, and treated with T-DM1. T-DM1 not bound to exosomes was removed using HER2-coated magnetic beads. Exosome samples were analyzed by electron microscopy, flow cytometry and Western blotting. Binding of T-DM1-containing exosomes to cancer cells and T-DM1 internalization were investigated with confocal microscopy. Effects of T-DM1-containg exosomes on cancer cells were investigated with the AlamarBlue cell proliferation assay and the Caspase-Glo 3/7 caspase activation assay. RESULTS: T-DM1 binds to exosomes derived from HER2-positive cancer cells, but not to exosomes derived from HER2-negative MCF-7 cells. HER2-positive SKBR-3 cells accumulated T-DM1 after being treated with T-DM1-containg exosomes, and treatment of SKBR-3 and EFM-192A cells with T-DM1-containing exosomes resulted in growth inhibition and activation of caspases 3 and/or 7. CONCLUSION: T-DM1 binds to exosomes derived from HER2-positive cancer cells, and T-DM1 may be carried to other cancer cells via exosomes leading to reduced viability of the recipient cells. The results suggest a new mechanism of action for T-DM1, mediated by exosomes derived from HER2-positive cancer.

Transmembrane TNF-α Reverse Signaling Inhibits Lipopolysaccharide-Induced Proinflammatory Cytokine Formation in Macrophages by Inducing TGF-ß: Therapeutic Implications.

TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α-bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti-TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-ß. Based on inhibitory experiments, the production of TGF-ß1 is regulated via Jun kinases, whereas that of other TGF-ßs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-ß by Abs prevented the mTNF-α-mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-ß. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-ß, we show that they do not use the mTNF-α signaling pathway. Because TGF-ß possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-ß synthesis by those TNF-α-targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn's disease, Wegener's granulomatosis, or sarcoidosis. Additionally, none of the TNF-α-targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling.

OBJECTIVES: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. MATERIAL AND METHODS: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the "dot-type" (representing late G2 phase) and "mitotic" immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. RESULTS: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G2 phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G2 phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G2 staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G2/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). CONCLUSIONS: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G2 phase B cells in reactive folliculi directs to physiological G2/M checkpoint blockade. In contrast, accelerated G2/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

Limbal and Conjunctival Epithelial Cell Cultivation on Contact Lenses-Different Affixing Techniques and the Effect of Feeder Cells.

OBJECTIVES: Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells. METHODS: Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells. RESULTS: Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate. CONCLUSIONS: Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.

TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity.

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

CD1d favors MHC neighborhood, GM1 ganglioside proximity and low detergent sensitive membrane regions on the surface of B lymphocytes.

BACKGROUND: Cluster of differentiation 1 (CD1) represents a family of proteins which is involved in lipid-based antigen presentation. Primarily, antigen presenting cells, like B cells, express CD1 proteins. Here, we examined the cell-surface distribution of CD1d, a subtype of CD1 receptors, on B lymphocytes. METHODS: Fluorescence labeling methods, including fluorescence resonance energy transfer (FRET),were employed to investigate plasma membrane features of CD1d receptors. RESULTS: High FRET efficiency was observed between CD1d and MHC I heavy chain (MHC I-HC), ß2-microglobulin(ß2m) and MHC II proteins in the plasma membrane. In addition, overexpression of CD1d reduced the expression of MHC II and increased the expression of MHC I-HC and ß2m proteins on the cell-surface. Surprisingly, ß2m dependent CD1d isoform constituted only ~15% of the total membrane CD1d proteins. Treatment of B cells with methyl-ß-cyclodextrin (MßCD) / simvastatin caused protein rearrangement; however, FRET demonstrated only minimal effect of these chemicals on the association between CD1d and GM1 ganglioside on cell-surface.Likewise, a modest effect was only observed in a co-culture assay between MßCD/simvastatin treated C1R­CD1d cells and invariant natural killer T cells on measuring secreted cytokines (IFNγ and IL4). Furthermore,CD1d rich regions were highly sensitive to low concentration of Triton X-100. Physical proximity between CD1d, MHC and GM1 molecules was also detected in the plasma membrane. CONCLUSIONS: An intricate relationship between CD1d, MHC, and lipid species was found on the membrane of human B cells. GENERAL SIGNIFICANCE: Organization of CD1d on the plasma membrane might be critical for its biological functions.

Understanding FRET as a research tool for cellular studies.

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1-10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

Potentiation of inhibitory synaptic transmission by extracellular ATP in rat suprachiasmatic nuclei.

The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.

Function of RasGRP3 in the formation and progression of human breast cancer.

INTRODUCTION: Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. METHODS: The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. RESULTS: RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells. CONCLUSIONS: Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.

Galactosyltransferases from Arabidopsis thaliana in the biosynthesis of type II arabinogalactan: molecular interaction enhances enzyme activity.

BACKGROUND: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood. RESULTS: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses ß-1,6-galactosyltransferase activity, elongating ß-1,6-galactan side chains and forming 6-Gal branches on the ß-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Förster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of ß-1,6-galactosyltransferase activities compared to AtGALT29A alone. CONCLUSIONS: AtGALT29A is a ß-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to ß-1,6-galactan and to ß-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.

Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.