BBA - Molecular Cell Research (v.1542, #1-3)

Multiple roles of the invariant chain in MHC class II function by Pamela Stumptner-Cuvelette; Philippe Benaroch (1-13).
Keywords: Class II associated invariant chain; Protein trafficking;

The involvement of NF-κB in the constitutive overexpression of cyclooxygenase-2 in cyclooxygenase-1 null cells by Takuro Kanekura; Sarita Goorha; Kanyawim Kirtikara; Leslie R. Ballou (14-22).
We recently reported that there was enhanced cyclooxygenase (COX)-2 expression and prostaglandin E2 biosynthesis in COX-1 deficient (COX-1−/−) cells. We also observed that the growth of COX-1−/− cells was significantly retarded compared to wild-type (WT) and COX-2 deficient (COX-2−/−) cells. In this study, COX-2 expression and its promoter activity were compared in immortalized, nontransformed fibroblasts from WT, COX-1−/− or COX-2−/− mice in the context of the role of COX-2 as a growth regulator. When compared with WT cells expressing both COX isoenzymes, constitutive COX-2 protein and promoter activity were significantly higher in COX-1−/− cells as determined by Western blotting and luciferase assays using a 5′-flanking promoter construct of the murine COX-2 gene. The luciferase assay using a series of luciferase-linked COX-2 promoter deletions transfected into COX-1−/− cells indicated that a region involving NF-κB plays a significant role in regulating constitutive COX-2 expression. Data from electrophoretic mobility shift assays showed that COX-1−/− cells contained higher levels of activated NF-κB than either WT or COX-2−/− cells. Furthermore, COX-2 promoter activity was significantly inhibited by the oligonucleotides (ODNs) containing the NF-κB element (NF-κB decoy ODNs) but not by the scrambled control ODNs, as examined by the luciferase assay. These findings indicate that constitutive COX-2 promoter activity and protein expression are enhanced in COX-1−/− fibroblasts and that signaling via the NF-κB pathway is involved in the transcriptional control of constitutive COX-2 expression.
Keywords: Cyclooxygenase-1 null cell; Cyclooxygenase-2; Nuclear factor κB; Cyclooxygenase-2 promoter regulation;

We have investigated EGF-driven signaling processes in rat intestinal epithelial cell lines that overexpress either the α5β1 integrin or the α2β1 integrin. Both cell types display efficient activation of Erk/MAP kinase, but only the α5β1 expressing cells display a strong activation of Akt. A complex is formed between activated EGFR and α5β1, but not with α2β1; this complex also contains ErbB3 and p85. Thus α5β1 can support efficient activation of both the Erk and the phosphatidylinositol-3-kinase/Akt branches of the EGFR signaling cascade, whereas α2β1 can support only the Erk branch.
Keywords: Integrin; Phosphatidylinositol-3-kinase; Akt/PKB; Epidermal growth factor receptor; Signaling;

Novobiocin is a novel inducer of CD38 on cells of the myelomonocytic lineage by Andrea Thiele; Martin Pfister; Monika Erbes; Michael Cross; Maria Hänsch; Sunna Hauschildt (32-40).
KG-1a, HL-60 and U-937 cells, which represent different stages of myelopoiesis, showed growth retardation in response to the coumarin antibiotic novobiocin. Novobiocin was found to increase CD38 expression (in all three cell lines) and to induce differentiation along the monocytic path in HL-60 and U-937 cells but not in KG-1a cells. The increase in surface expression of CD38 was matched by NAD glycohydrolase activity and by increases in the level of specific mRNA, indicating that the gene product is active and that regulation occurs at the level of transcription or mRNA stability. Of the three cell lines tested, only the early KG-1a expressed N-cadherin, a member of Ca2+-dependent adhesion molecules involved in embryonic differentiation processes. In contrast to CD38, N-cadherin was slightly down-regulated pointing to a specific role of novobiocin in gene regulation.
Keywords: CD38; Novobiocin; Myelomonocytic cell line; Differentiation; N-Cadherin;

Characterization of ubiquilin 1, an mTOR-interacting protein by Shilan Wu; Alexei Mikhailov; Heidi Kallo-Hosein; Kenta Hara; Kazuyoshi Yonezawa; Joseph Avruch (41-56).
The mTOR protein kinase is known to control cell cycle progression and cell growth through regulation of translation, transcription, membrane traffic and protein degradation. Known interactions of mTOR do not account for the multiple functions of this protein. Using a non-catalytic segment of mTOR (1–670) as bait in a yeast two-hybrid screen for interacting proteins, ubiquilin 1 (NM013438) was identified. Ubiquilin 1 is a member of a phylogenetically conserved gene family of unknown function, characterized by an N-terminal ubiquitin-like (Ubq) domain, a C-terminal ubiquitin associated (Uba) domain and a central region containing numerous NPXϕ motifs (X, any; ϕ, hydrophobic amino acid). GST-ubiquilin 1 binds specifically to FLAG-mTOR (residues 1–670) in mammalian cells; residues 570–670 of mTOR and 226–323 of ubiquilin 1 are required for this interaction. Both mTOR and ubiquilin immunoreactivity appear as fine speckles throughout the cytoplasm; significant colocalization with cytoskeletal elements, early endosomes or proteasomes is not observed. As assessed by cell fractionation, mTOR is predominantly associated with low density membranes, along with 10% of ubiquilin 1. Ubiquilin 1 is a rapamycin-insensitive phosphoprotein. Overexpression of ubiquilin 1 does not alter the kinase activity of cotransfected mTOR or the phosphorylation of the mTOR target, p70 S6 kinase, in the presence or absence of rapamycin. Our data suggest that we have identified a novel mTOR interactor, ubiquilin 1. The biological significance of this, presumably membrane based, interaction, requires further study.
Keywords: mTOR; Rapamycin; Ubiquilin 1; p70 S6 kinase;

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by αMSH and ornithine decarboxylase inhibition by J. Sánchez Mas; M. Martínez-Esparza; C.M. Bastida; F. Solano; R. Peñafiel; J.C. García-Borrón (57-65).
Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by α-melanocyte-stimulating hormone (αMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, αMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or αMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor α-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with αMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to αMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by αMSH.
Keywords: Melanoma cell; Tyrosinase; Ornithine decarboxylase; α-Difluoromethylornithine; Polyamine; Regulation of melanogenesis;

Tumor necrosis factor-α exerts interleukin-6-dependent and -independent effects on cultured skeletal muscle cells by Belén Alvarez; LeBris S Quinn; Sı́lvia Busquets; Maria T Quiles; Francisco J López-Soriano; Josep M Argilés (66-72).
In vivo studies have shown that cancer-associated skeletal muscle wasting (cachexia) is mediated by two cytokines, tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). It has been unclear from these studies whether TNF exerts direct effects on skeletal muscle and/or whether these effects are mediated via IL-6. Previous studies from our laboratory have shown that TNF induces IL-6 mRNA expression in cultured skeletal muscle cells. To further investigate the relationship between TNF and IL-6, the effects of TNF and IL-6 on protein and DNA dynamics in murine C2C12 skeletal myotube cultures were determined. At 1000 U/ml, TNF induced 30% increases in protein and DNA content. The effects of TNF on protein accumulation were inhibited by aphidicolin, an inhibitor of DNA synthesis. IL-6 mimicked the effects of TNF on C2C12 cultures, inducing a 32% increase in protein accumulation and a 71% increase in the rate of protein synthesis. IL-6 also decreased expression of mRNA for several proteolytic system components, including ubiquitin 2.4 kb (51%) and 1.2 kb (63%), cathepsin B (39%) and m-calpain (47%), indicating that IL-6 acts on both protein synthesis and degradation. Incubation of murine C2C12 myotube cultures with TNF (1000 U/ml) in the presence of a polyclonal mouse anti-IL-6 antibody resulted in an abolishment of the effects of TNF on protein synthesis, but did not inhibit TNF-induced stimulation of DNA synthesis. These findings indicate that the effects of TNF on muscle protein synthesis are mediated by IL-6, but that TNF exerts IL-6-independent effects on proliferation of murine skeletal myoblasts.
Keywords: Tumor necrosis factor α; Skeletal muscle; Interleukin-6; Cytokine;

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser180) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser180 is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [32P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.
Keywords: cAMP; cAMP-dependent protein kinase; Autophosphorylation; Candida albicans;

Oxidative stress induces apoptosis in liver parenchymal cells. The present study demonstrates that the substitution of fructose for glucose as sole carbon source in the incubation medium reduced apoptosis due to reoxygenation up to 50% in cultured rat hepatocytes. This anti-apoptotic action of fructose cannot be explained by the effects of this sugar on the intracellular ATP concentration and the ATP/ADP ratio. Rather, the suppression of apoptosis by fructose seems to be a consequence of remarkably higher intracellular levels of glutathione observed during reoxygenation in fructose-fed hepatocytes in contrast to glucose-fed ones. With fructose as substrate, the generation of excess reactive oxygen species (ROS) during the initial phase of reoxygenation was strongly reduced. With respect to ROS reduction and stabilization of the cellular glutathione pool fructose was found as efficient as a pretreatment of glucose fed cells with N-acetyl-l-cysteine. The enhanced metabolization of ROS by the glutathione/glutathione peroxidase system in fructose-cultured hepatocytes under reoxygenation was expected to improve their mitochondrial status so that late events in the apoptotic pathway are suppressed. This could be confirmed by the reduced release of cytochrome c from mitochondria into the cytosol as well as by the observed decrease of caspase-3 activity during reoxygenation.
Keywords: Rat hepatocyte; Apoptosis; Fructose; Oxidative stress; Glutathione; Reactive oxygen species;

Inhibition of somatostatin receptor 5-signaling by mammalian regulators of G-protein signaling (RGS) in yeast by Janice L. Kong; Rosemarie Panetta; Wei Song; Wendy Somerville; Michael T. Greenwood (95-105).
Regulators of G-protein signaling (RGSs) are negative regulators of G-protein coupled receptor (GPCR)-mediated signaling that function to limit the lifetime of receptor-activated Gα-proteins. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1–LacZ reporter gene by the STE2 encoded GPCR in yeast. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SST5) in yeast. In the absence of RGSs, FUS1–LacZ activation in response to somatostatin increased in a dose-dependent manner in cells expressing SST5. In contrast to the results obtained with Ste2p, all RGSs completely inhibited SST5-mediated signaling even at concentrations of agonist as high as 10−5 M. The ability of RGSs to inhibit SST5 signaling was further assessed in cells expressing modified Gpa1 proteins. Even though SST5-mediated FUS1–LacZ activation was 5-fold more efficient with a Gpa1p/Gi3α chimera, response to somatostatin was completely abolished by all four RGSs. Furthermore, we demonstrate that RGS1, RGS2 and RGS5 have reduced ability to inhibit SST5-mediated activation of the RGS-resistant Gpa1pGly302Ser mutant suggesting that the ability to interact with the Gα-protein is required for the inhibition of signaling. Taken together, our results indicate that RGSs serve as better GAPs for Gpa1p when activated by SST5 than when this G-protein is activated by Ste2p.
Keywords: Regulator of G-protein signaling (RGS); Heterologous expression; G-protein coupled receptor; Signal transduction;

Infection of cells with replication deficient adenovirus induces cell cycle alterations and leads to downregulation of E2F-1 by Hartmut Kuhn; Uta Liebers; Christian Gessner; Leonid Karawajew; Velia Ruppert; Axel Schumacher; Christian Witt; Gerhard Wolff (106-115).
Gene products of recombinant replication-deficient adenovirus vectors of the first generation (Ad vector) can induce cell cycle dysregulation and apoptosis after infection in eukaryotic cells. The mechanisms underlying this complex process are largely unknown. Therefore, we investigated the regulation of the pRb/E2F-1 complex, which controls transition from G0/G1 to S phase of the cell cycle. As Ad vector infection results in a decrease in the number of cells in G0/G1 phase of the cell cycle, we observed a decline of the pRb protein level and, surprisingly, also a decrease of the E2F-1 protein and mRNA level in infected cell lines. Furthermore, in contrast to the reduction of cells in the G0/G1 phase we observed increased protein levels of p53 and p21 proteins. However, as experiments in p53 deficient cell lines indicated, the decrease of pRb and E2F-1 is independent of p53 and p21 expression. Moreover, results obtained with Rb deficient cell lines indicated that the reduced E2F-1 expression is independent of pRb. These results suggest that Ad vector-induced cell cycle dysregulation is associated with a specific downregulation of E2F-1 independent of Rb and p53 genomic status of cells.
Keywords: Adenovirus vector; pRb; E2F-1; p53; Cell cycle;

Expression and localization of AQP5 in the stomach and duodenum of the rat by Most.Nahid Parvin; Keiko Tsumura; Tetsuya Akamatsu; Norio Kanamori; Kazuo Hosoi (116-124).
The expression, localization, and regulation of aquaporin 5 (AQP5), a member of the water channel family of proteins, was investigated in tissues of the rat gastrointestinal tract. Reverse transcriptase–polymerase chain reaction (RT–PCR) detected AQP5 mRNA in the lower stomach and duodenum. DNA sequencing confirmed that the cDNA fragment amplified had the complete sequence of the AQP5 cDNA fragment. Western blot analysis indicated the expression of a 27 kDa molecular mass AQP5 protein in the lower stomach and duodenum, which size was the same as that found for the protein in the submandibular gland and lungs. By immunohistochemistry using the IgG affinity-purified AQP5 antibody, the pyloric gland and Brunner’s gland were primarily stained in the lower stomach and duodenum, respectively; a strong staining appeared in the apical and lateral membranes in both glands. These results indicate that AQP5 is present in the rat lower stomach and duodenum where it may be involved in a water transport mechanism. These results also support the idea that AQP5, and probably other aquaporins, are involved in water secretion in the stomach and duodenum although the volume of water transported via AQPs is unclear.
Keywords: Aquaporin 5; Water channel; Lower stomach; Duodenum; Pyloric gland; Brunner’s gland;

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein by Anna Maria Porcelli; Katia Scotlandi; Rosaria Strammiello; Gabriella Gislimberti; Nicola Baldini; Michela Rugolo (125-138).
The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2′,7′-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl and Na+ gradients, both in HEPES- and HCO3 /CO2-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4′-diisothiocyanate dihydrostilbene-2,2′-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na+/H+ exchanger. In addition to the amiloride-sensitive Na+/H+ exchanger, another H+ extruding mechanism was detected in U-2 OS cells, the Na+-dependent HCO3 /Cl exchanger. No significant difference in resting pHi and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V max and K′ values for intracellular [H+] of the Na+/H+ exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pHi regulation in U-2 OS cells, the kinetic parameters of the Na+/H+ exchanger were altered, suggesting a functional relationship between the two membrane proteins.
Keywords: P-glycoprotein; MDR1 gene; Intracellular pH; Na+/H+ exchanger; Human osteosarcoma cell;

Apoptosis of thymic cells induced by glucocorticoids (GC) and T-cell receptor (TCR) engagement are mutually antagonistic. We demonstrate that cAMP enhances GC and antagonizes TCR (anti-CD3) apoptosis on the same cell (DO-11.10 and 2B4.11 T-cell hybridomas). We analyzed the activity of several transcription factors in this cAMP dual, stimulus-dependent, regulatory action. Anti-CD3 increases kB-activity which is inhibited by CPTcAMP or dexamethasone (DEX), supporting the proapoptotic role of NFkB on TCR-induced apoptosis. Anti-CD3 not only increases kB- but diminishes GC response element (GRE)-activity induced by DEX, suggesting that TCR-mediated blockade of GC-induced apoptosis involves not only the proposed antiapoptotic action of NF-kB on GC, but also the inhibition of GRE-regulated proapoptotic genes. To test the involvement of CRE-driven transcription in the cAMP dual apoptotic regulation, cells were transfected with a CRE decoy DNA oligomer. Blockade of CRE transactivation with decoy targeting of CRE completely blocked the protection of TCR-induced apoptosis by cAMP, while it did not modify the enhancement by cAMP on GC-induced apoptosis. We show that CRE-binding factors have a definite role in T-cell apoptosis: they are involved in cAMP protection of TCR- but not in cAMP potentiation of GC-induced apoptosis.
Keywords: Glucocorticoids; Cyclic AMP; T cell receptor; cAMP responsive element binding protein; Apoptosis;

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols by Julie Jodoin; Michel Demeule; Richard Béliveau (149-159).
Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 μg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CHRC5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CHRC5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.
Keywords: Green tea polyphenol; P-Glycoprotein; Catechin; Multidrug resistance; Epigallocatechin gallate;

Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii by Mary Q. Stewart; Ralf van Dijk; Marten Veenhuis; Joel M. Goodman (160-172).
A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii. The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of ∼80–90 kDa. Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a ∼20 kDa species, suggesting it is an oligomer. The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC. Additionally, monomers of alcohol oxidase (AO), an abundant protein of C. boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease. Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.
Keywords: Peroxisome; Microbody; Serine protease; Alcohol oxidase; Candida boidinii;

Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum by Khadidja Haidara; Isabelle Morel; Valérie Abaléa; Marielle Gascon Barré; Francine Denizeau (173-185).
The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4–0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.
Keywords: Oxidative stress; Apoptosis; Cytochrome c; Bcl-2; Bax; Relocalization;

Shock wave-mediated molecular delivery into cells by Tetsuya Kodama; Apostolos G. Doukas; Michael R. Hamblin (186-194).
A single shock wave generated by a shock tube is able to effectively deliver macromolecules such as fluorescein isothiocyanate–dextran into the cytoplasm of living cells without causing cytotoxicity. We report on the effect of varying the molecular weight of the dextran and the number of shock waves on the efficiency of delivery into a cancer cell line. The fraction of cells permeabilized and the total fluorescence delivered were measured by flow cytometry, and the cellular viability by a tetrazolium assay on adherent cells and these values were compared to cell permeabilization using digitonin. Shock waves can deliver molecules of up to 2 000 000 molecular weight into the cytoplasm of cells without toxicity and may have applications in gene therapy.
Keywords: Shock tube; Membrane permeabilization; FITC-dextran; Digitonin; Flow cytometry; Fluorescence;

The ability of a soluble heparin-binding oligopeptide sequence derived from the von Willebrand factor (vWF) to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with six-fold decrease in cell–substrate adhesive strength (P<0.001), a biphasic effect on migration speed (P<0.05), as well as a dose-dependent reduction in the percentage of motile cells and the cell dispersion coefficient (μ=S 2 T/2). The specificity of this response to the vWF-derived heparin-binding peptide was supported by the absence of an observed effect in the presence of either a scrambled peptide or a consensus heparin-binding peptide sequence of similar heparin affinity. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptide domains located in soluble peptide fragments may modulate chemokinetic cell migration behavior and other adhesion-related processes.
Keywords: Heparan sulfate; Chemokinesis; Cell adhesion; Focal contact; Extracellular matrix;

Green tea polyphenol (−)-epigallocatechin 3-gallate inhibits MMP-2 secretion and MT1-MMP-driven migration in glioblastoma cells by Borhane Annabi; Marie-Paule Lachambre; Nathalie Bousquet-Gagnon; Martine Pagé; Denis Gingras; Richard Béliveau (209-220).
We have recently shown that green tea polyphenols, and especially (−)-epigallocatechin 3-gallate (EGCg), acted as potent inhibitors of matrix metalloproteinase activities as well as of proMMP-2 activation (M. Demeule, M. Brossard, M. Page, D. Gingras, R. Beliveau, Biochim. Biophys. Acta 1478 (2000)). In the present work, we sought to examine the involvement of MT1-MMP in the EGCg-induced inhibition of proMMP-2 activation. The incubation of U-87 glioblastoma cells in the presence of concanavalin A or cytochalasin D, two potent activators of MT1-MMP, resulted in proMMP-2 activation that was correlated with the cell surface proteolytic processing of MT1-MMP to its inactive 43 kDa form. Addition of EGCg strongly inhibited the MT1-MMP-dependent proMMP-2 activation. The inhibitory effect of EGCg on MT1-MMP was also demonstrated by the down-regulation of MT1-MMP transcript levels and by the inhibition of MT1-MMP-driven cell migration of transfected COS-7 cells. These observations suggest that this catechin may act at both the MT1-MMP gene and protein expression levels. In addition, treatment of cells with non-cytotoxic doses of EGCg significantly reduced the amount of secreted proMMP-2, and led to a concomitant increase in intracellular levels of that protein. This effect was similar to that observed using well-characterized secretion inhibitors such as brefeldin A and manumycin, suggesting that EGCg could also potentially act on intracellular secretory pathways. Taken together, these results indicate that EGCg targets multiple MMP-mediated cellular events in cancer cells and provides a new mechanism for the anticancer properties of that molecule.
Keywords: Angiogenesis; Cancer; Glioblastoma; Green tea; Membrane-type 1 matrix metalloproteinase;

Author Index (221-222).

Cumulative Contents (223-224).