19 results on '"Klarenbeek, Jeffrey"'
Search Results
2. Dynamic FRET-FLIM based screening of signal transduction pathways
- Author
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Harkes, Rolf, Kukk, Olga, Mukherjee, Sravasti, Klarenbeek, Jeffrey, van den Broek, Bram, and Jalink, Kees
- Published
- 2021
- Full Text
- View/download PDF
3. Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells
- Author
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Vink, Stefan R., van der Luit, Arnold H., Klarenbeek, Jeffrey B., Verheij, Marcel, and van Blitterswijk, Wim J.
- Published
- 2007
- Full Text
- View/download PDF
4. TRPM7 residue S1269 mediates cAMP dependence of Ca2+ influx.
- Author
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Broertjes, Jorrit, Klarenbeek, Jeffrey, Habani, Yasmin, Langeslag, Michiel, and Jalink, Kees
- Subjects
- *
CALCIUM ions , *TRP channels , *BREAST cancer , *CANCER relapse , *CYCLIC adenylic acid - Abstract
The nonspecific divalent cation channel TRPM7 (transient receptor potential-melastatin-like 7) is involved in many Ca2+ and Mg2+-dependent cellular processes, including survival, proliferation and migration. TRPM7 expression predicts metastasis and recurrence in breast cancer and several other cancers. In cultured cells, it can induce an invasive phenotype by promoting Ca2+-mediated epithelial-mesenchymal transition. We previously showed that in neuroblastoma cells that overexpress TRPM7 moderately, stimulation with Ca2+-mobilizing agonists leads to a characteristic sustained influx of Ca2+. Here we report that sustained influx through TRPM7 is abruptly abrogated by elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Using pharmacological inhibitors and overexpression studies we show that this blockage is mediated by the cAMP effector Protein Kinase A (PKA). Mutational analysis demonstrates that the Serine residue S1269, which is present proximal to the coiled-coil domain within the protein c-terminus, is responsible for sensitivity to cAMP. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
5. Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation.
- Author
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Argenzio, Elisabetta, Klarenbeek, Jeffrey, Kedziora, Katarzyna M., Nahidiazar, Leila, Isogai, Tadamoto, Perrakis, Anastassis, Jalink, Kees, Moolenaar, Wouter H., and Innocenti, Metello
- Subjects
- *
PROFILIN , *RHO GTPases , *INTRACELLULAR calcium , *CELLULAR signal transduction , *POLYMERIZATION , *CELL adhesion - Abstract
Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
6. Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity.
- Author
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Klarenbeek, Jeffrey, Goedhart, Joachim, van Batenburg, Aernoud, Groenewald, Daniella, and Jalink, Kees
- Subjects
- *
CYCLIC-AMP-dependent protein kinase , *SIGNAL-to-noise ratio , *CYTOLOGY , *GENETIC mutation , *FLUOROPHORES , *FLUORESCENCE - Abstract
Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 μM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark) acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
7. TRPM7 triggers Ca2+ sparks and invadosome formation in neuroblastoma cells.
- Author
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Visser, Daan, Langeslag, Michiel, Kedziora, Katarzyna M., Klarenbeek, Jeffrey, Kamermans, Alwin, Horgen, F. David, Fleig, Andrea, van Leeuwen, Frank N., and Jalink, Kees
- Abstract
Abstract: Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca
2+ signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much attention as a regulator of cell adhesion, migration and (localized) Ca2+ signaling. Overexpression and knockdown of TRPM7 affects actomyosin contractility and the formation of cell adhesions such as invadosomes and focal adhesions, but the role of TRPM7-mediated Ca2+ signals herein is currently not understood. Using Total Internal Reflection Fluorescence (TIRF) Ca2+ fluorometry and a novel automated analysis routine we have addressed the role of Ca2+ in the control of invadosome dynamics in N1E-115 mouse neuroblastoma cells. We find that TRPM7 promotes the formation of highly repetitive and localized Ca2+ microdomains or “Ca2+ sparking hotspots” at the ventral plasma membrane. Ca2+ sparking appears strictly dependent on extracellular Ca2+ and is abolished by TRPM7 channel inhibitors such as waixenicin-A. TRPM7 inhibition also induces invadosome dissolution. However, invadosome formation is (functionally and spatially) dissociated from TRPM7-mediated Ca2+ sparks. Rather, our data indicate that TRPM7 affects actomyosin contractility and invadosome formation independent of Ca2+ influx. [Copyright &y& Elsevier]- Published
- 2013
- Full Text
- View/download PDF
8. A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range.
- Author
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Klarenbeek, Jeffrey B., Goedhart, Joachim, Hink, Mark A., Gadella, Theodorus W. J., and Jalink, Kees
- Subjects
- *
DETECTORS , *CARRIER proteins , *SIGNAL-to-noise ratio , *FLUORESCENCE , *INFORMATION measurement , *PROTEIN binding , *QUANTUM chemistry , *QUANTUM efficiency - Abstract
FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM) or by Sensitized Emission (SE), e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed TEpacVV, which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that TEpacVV appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, TEpacVV should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
9. Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors.
- Author
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Leyton-Puig, Daniela, Isogai, Tadamoto, Argenzio, Elisabetta, van den Broek, Bram, Klarenbeek, Jeffrey, Janssen, Hans, Jalink, Kees, and Innocenti, Metello
- Abstract
Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
10. TRPM7 controls mesenchymal features of breast cancer cells by tensional regulation of SOX4.
- Author
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Kuipers, Arthur J., Middelbeek, Jeroen, Vrenken, Kirsten, Pérez-González, Carlos, Poelmans, Geert, Klarenbeek, Jeffrey, Jalink, Kees, Trepat, Xavier, and van Leeuwen, Frank N.
- Subjects
- *
TRP channels , *MESENCHYMAL stem cells , *CANCER cells , *BREAST cancer , *GENETIC regulation , *TRANSCRIPTION factors , *METASTASIS , *GENE expression - Abstract
Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo . Here, we show that TRPM7 contributes to maintaining a mesenchymal phenotype in breast cancer cells by tensional regulation of the EMT transcription factor SOX4. The functional consequences of SOX4 knockdown closely mirror those produced by TRPM7 knockdown. By traction force measurements, we demonstrate that TRPM7 reduces cytoskeletal tension through inhibition of myosin II activity. Moreover, we show that SOX4 expression and downstream mesenchymal markers are inversely regulated by cytoskeletal tension and matrix rigidity. Overall, our results identify SOX4 as a transcription factor that is uniquely sensitive to cellular tension and indicate that TRPM7 may contribute to breast cancer progression by tensional regulation of SOX4. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
11. Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors.
- Author
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Kukk O, Klarenbeek J, and Jalink K
- Subjects
- Microscopy, Fluorescence methods, Photons, Second Messenger Systems, Fluorescence Resonance Energy Transfer methods, Optical Imaging
- Abstract
Second messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-efficient FLIM instrumentation, it is becoming the method of choice for FRET detection in signaling studies. Here, we describe a detailed protocol for time domain FLIM, using the EPAC-based FRET sensor to measure changes in cellular cAMP levels with high spatiotemporal resolution as an example., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
12. TRPM7 residue S1269 mediates cAMP dependence of Ca2+ influx.
- Author
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Broertjes J, Klarenbeek J, Habani Y, Langeslag M, and Jalink K
- Subjects
- Animals, Calcium Signaling drug effects, Cell Line, Tumor, Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors, Isoquinolines pharmacology, Mice, Neurons drug effects, Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Sulfonamides pharmacology, Calcium metabolism, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Neurons metabolism, TRPM Cation Channels metabolism
- Abstract
The nonspecific divalent cation channel TRPM7 (transient receptor potential-melastatin-like 7) is involved in many Ca2+ and Mg2+-dependent cellular processes, including survival, proliferation and migration. TRPM7 expression predicts metastasis and recurrence in breast cancer and several other cancers. In cultured cells, it can induce an invasive phenotype by promoting Ca2+-mediated epithelial-mesenchymal transition. We previously showed that in neuroblastoma cells that overexpress TRPM7 moderately, stimulation with Ca2+-mobilizing agonists leads to a characteristic sustained influx of Ca2+. Here we report that sustained influx through TRPM7 is abruptly abrogated by elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Using pharmacological inhibitors and overexpression studies we show that this blockage is mediated by the cAMP effector Protein Kinase A (PKA). Mutational analysis demonstrates that the Serine residue S1269, which is present proximal to the coiled-coil domain within the protein c-terminus, is responsible for sensitivity to cAMP., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
- Full Text
- View/download PDF
13. Recording intracellular cAMP levels with EPAC-based FRET sensors by fluorescence lifetime imaging.
- Author
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Raspe M, Klarenbeek J, and Jalink K
- Subjects
- Biosensing Techniques instrumentation, Cell Line, Tumor, Fluorescence Resonance Energy Transfer methods, Genes, Reporter, Humans, Microscopy, Fluorescence methods, Biosensing Techniques methods, Cyclic AMP metabolism, Microscopy, Fluorescence instrumentation
- Abstract
Eukaryotic cells use second messengers such as Ca(2+), IP3, cGMP, and cAMP to transduce extracellular signals like hormones, via membrane receptors to downstream cellular effectors. FRET-based sensors are ideal to visualize and measure these rapid changes of second messenger concentrations in time and place. Here, we describe the use of EPAC-based FRET sensors to measure cAMP with spatiotemporal resolution in cells by fluorescence lifetime imaging (FLIM).
- Published
- 2015
- Full Text
- View/download PDF
14. TRPM7 triggers Ca2+ sparks and invadosome formation in neuroblastoma cells.
- Author
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Visser D, Langeslag M, Kedziora KM, Klarenbeek J, Kamermans A, Horgen FD, Fleig A, van Leeuwen FN, and Jalink K
- Subjects
- Acetates pharmacology, Actomyosin metabolism, Animals, Cell Adhesion drug effects, Cell Line, Tumor, Cell Membrane metabolism, Cell Movement drug effects, Diterpenes pharmacology, Mice, Neuroblastoma metabolism, Neuroblastoma pathology, RNA Interference, RNA, Small Interfering metabolism, TRPM Cation Channels antagonists & inhibitors, TRPM Cation Channels genetics, Calcium metabolism, Calcium Signaling, TRPM Cation Channels metabolism
- Abstract
Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca2+ signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much attention as a regulator of cell adhesion, migration and (localized) Ca2+ signaling. Overexpression and knockdown of TRPM7 affects actomyosin contractility and the formation of cell adhesions such as invadosomes and focal adhesions, but the role of TRPM7-mediated Ca2+ signals herein is currently not understood. Using Total Internal Reflection Fluorescence (TIRF) Ca2+ fluorometry and a novel automated analysis routine we have addressed the role of Ca2+ in the control of invadosome dynamics in N1E-115 mouse neuroblastoma cells. We find that TRPM7 promotes the formation of highly repetitive and localized Ca2+ microdomains or "Ca2+ sparking hotspots" at the ventral plasma membrane. Ca2+ sparking appears strictly dependent on extracellular Ca2+ and is abolished by TRPM7 channel inhibitors such as waixenicin-A. TRPM7 inhibition also induces invadosome dissolution. However, invadosome formation is (functionally and spatially) dissociated from TRPM7-mediated Ca2+ sparks. Rather, our data indicate that TRPM7 affects actomyosin contractility and invadosome formation independent of Ca2+ influx., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
15. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons.
- Author
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Polito M, Klarenbeek J, Jalink K, Paupardin-Tritsch D, Vincent P, and Castro LR
- Abstract
The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-S(H150)) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 MSNs. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.
- Published
- 2013
- Full Text
- View/download PDF
16. Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells.
- Author
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Alderliesten MC, Klarenbeek JB, van der Luit AH, van Lummel M, Jones DR, Zerp S, Divecha N, Verheij M, and van Blitterswijk WJ
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Tumor, DNA Damage, Down-Regulation, Extracellular Signal-Regulated MAP Kinases metabolism, Fas Ligand Protein metabolism, Inositol Polyphosphate 5-Phosphatases, Lymphoma pathology, Mice, Phosphatidylinositol Phosphates metabolism, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases, Proto-Oncogene Proteins c-akt metabolism, Transferases (Other Substituted Phosphate Groups) metabolism, Phospholipid Ethers pharmacology, Phosphoric Monoester Hydrolases metabolism
- Abstract
S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.
- Published
- 2011
- Full Text
- View/download PDF
17. Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency.
- Author
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van Blitterswijk WJ, Klarenbeek JB, van der Luit AH, Alderliesten MC, van Lummel M, and Verheij M
- Subjects
- Animals, Apoptosis drug effects, CASP8 and FADD-Like Apoptosis Regulating Protein genetics, CASP8 and FADD-Like Apoptosis Regulating Protein metabolism, Cell Line, Tumor, Cell Proliferation drug effects, Down-Regulation, Fas Ligand Protein pharmacology, Fas-Associated Death Domain Protein genetics, Fas-Associated Death Domain Protein metabolism, Flow Cytometry, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Immunoblotting, Leupeptins pharmacology, Lymphoma genetics, Lymphoma metabolism, Lymphoma pathology, Membrane Microdomains drug effects, Membrane Microdomains metabolism, Mice, Microscopy, Confocal, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors, RNA, Small Interfering genetics, Reverse Transcriptase Polymerase Chain Reaction, Sphingomyelins deficiency, Transfection, fas Receptor genetics, Drug Resistance, Neoplasm, Lysophospholipids pharmacology, Sphingomyelins metabolism, fas Receptor metabolism
- Abstract
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.
- Published
- 2009
- Full Text
- View/download PDF
18. A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells.
- Author
-
van der Luit AH, Vink SR, Klarenbeek JB, Perrissoud D, Solary E, Verheij M, and van Blitterswijk WJ
- Subjects
- Animals, Antineoplastic Agents chemistry, Cell Line, Tumor, Endocytosis drug effects, HeLa Cells, Humans, Mice, Phosphatidylcholines biosynthesis, Phospholipid Ethers chemistry, Phospholipid Ethers pharmacology, Phospholipids chemistry, Phosphorylcholine analogs & derivatives, Phosphorylcholine chemistry, Phosphorylcholine pharmacology, Sphingomyelins biosynthesis, Time Factors, Antineoplastic Agents pharmacology, Apoptosis drug effects, Lymphoma pathology, Membrane Microdomains drug effects, Phospholipids pharmacology
- Abstract
Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-beta-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)-mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy.
- Published
- 2007
- Full Text
- View/download PDF
19. Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts.
- Author
-
Van der Luit AH, Budde M, Zerp S, Caan W, Klarenbeek JB, Verheij M, and Van Blitterswijk WJ
- Subjects
- Androstenes pharmacology, Animals, Bridged-Ring Compounds pharmacology, Cholesterol analysis, Cholesterol metabolism, Down-Regulation, Endocytosis drug effects, Fatty Acids, Monounsaturated pharmacology, Gene Expression drug effects, Membrane Microdomains drug effects, Membrane Microdomains metabolism, Mice, Norbornanes, Phospholipid Ethers metabolism, RNA, Small Interfering pharmacology, Sphingomyelins analysis, Sphingomyelins metabolism, Thiocarbamates, Thiones pharmacology, Transferases (Other Substituted Phosphate Groups) metabolism, Tumor Cells, Cultured, Apoptosis drug effects, Membrane Microdomains chemistry, Phospholipid Ethers pharmacology, Transferases (Other Substituted Phosphate Groups) biosynthesis
- Abstract
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.
- Published
- 2007
- Full Text
- View/download PDF
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