Biomedical subjects

Christian Slomianny

Publications and source records attributed to Christian Slomianny.

16 recordsLinked to original sources

PubMed2007

Prolactin stimulates prostate cell proliferation by increasing endoplasmic reticulum content due to SERCA 2b over-expression.

Prolactin (PRL) has been shown to be involved in the differentiation and proliferation of numerous tissues, including the prostate gland. Moreover, variations in [Ca2+]ER (calcium concentration within the endoplasmic reticulum) may play a role in cell growth. However, few studies have focused on the regulation of calcium homoeostasis by prolactin. The present study evaluates the regulation of calcium pools as well as the possible role of [Ca2+]ER variations as a signal for growth modulation by PRL. We show that PRL stimulates the proliferation of normal SV40 immortalized epithelial prostate (PNT1A) cells with a maximum effect at a dose of 100 ng/ml. We also show that 100 ng/ml PRL increases the [Ca2+]ER when measured either by indirect quantification with Fura-2AM after application of 1 mM thapsigargin or by direct quantification with Mag-Fura-2AM within the endoplas-mic reticulum. Western blot analysis shows that the SERCA 2b (sarcoendoplasmic calcium ATPase 2b) is over-expressed in PNT1A cells treated with 100 ng/ml PRL for 24 h. A small inter-fering RNA SERCA 2a/b, used to down-regulate endogenous SERCA 2b expression, reduced both PNT1A cell proliferation and [Ca2+]ER. We thus identify [Ca2+]ER and SERCA 2b as protagonists in PRL-induced proliferation.

Calcium↗

PubMed2006

Proteomics exploration reveals that actin is a signaling target of the kinase Akt.

The serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: alpha -actinin, valosin-containing protein, inhibitor kappaB kinase, mortalin, tubulin beta, cytokeratin 8, actin, 14-3-3sigma, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt co-immunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling.

Actins↗

PubMed2006

Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment.

Seven strains of Bacillus cereus isolated from the environment and from patients with diarrheic symptoms were examined from two angles: their spore surface properties, and their ability to adhere to stainless steel and to resist a cleaning in place (CIP) procedure. Our results revealed significant differences in their morphology (size of exosporium, length and number of appendages), hydrophobic character and surface protein composition. Most of these proteins originated in the vegetative cell and were tightly bound to the external surface of the exosporium such as EA1 or alanine racemase. Spore adhesion properties also varied from strain to strain. The ability to adhere was higher when spores were surrounded by long appendages, while the largest spores displayed the least resistance to cleaning. These observations suggest that food processing line contamination might be due to a given type of strain with specific surface properties (long appendages and small exosporium), which would represent an increased threat under the milder processing conditions required by consumers (minimally heat-treated foods for example) and by legal requirements (to limit effluents caused by hygiene procedures). Elsewhere, no clear relationship of the strain characteristics to the clinical vs. foodborne strains could be established.

Bacillus cereus↗

PubMed2006

The valosin-containing protein (VCP) is a target of Akt signaling required for cell survival.

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappaB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

Adenosine Triphosphatases↗

PubMed2006

Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells.

One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca2+ is central to growth control, understanding the mechanisms of Ca2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of alpha1-adrenergic receptors (alpha1-AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca2+ entry (ACE) relied mostly on transient receptor potential canonical 6 (TRPC6) channels, whose silencing by antisense hybrid depletion decreased both hPCE cell proliferation and ACE. In contrast, ACE and related growth arrest associated with purinergic receptors (P2Y-R) stimulation involved neither TRPC6 nor NFAT. Our findings show that alpha1-AR signaling requires the coupled activation of TRPC6 channels and NFAT to promote proliferation of hPCE cells and thereby suggest TRPC6 as a novel potential therapeutic target.

Adenosine Triphosphate↗

PubMed2004

Amylopectin biogenesis and characterization in the protozoan parasite Toxoplasma gondii, the intracellular development of which is restricted in the HepG2 cell line.

The obligate intracellular protozoan Toxoplasma gondii belongs to the phylum Apicomplexa, which is composed of numerous parasites causing major diseases such as malaria, toxoplasmosis and coccidiosis. The life cycle of T. gondii involves developmental processes from one stage to another with both asexual and sexual parasitic forms. Throughout their life cycle, some apicomplexan parasites accumulate a crystalline storage polysaccharide analogous to amylopectin within the cytoplasm. In T. gondii, both the slowly dividing encysted bradyzoites and the sporozoites of the sexual stage contain a high number of amylopectin granules (AG), while the rapidly replicating tachyzoites are devoid of amylopectin. It is thought that this storage polysaccharide may represent an energy reserve that could fuel the transition from one developmental stage to another one. At present, by comparison to glycogen and plant starch, little is known about the biosynthesis, structure and biological functions of amylopectin in T. gondii. Here, we describe an in vitro system allowing the production and purification of a large amount of amylopectin, which has been subjected to detailed biochemical and structural analyses. Our data indicate that T. gondii synthesizes a genuine amylopectin following changes in the environmental conditions and that this storage polysaccharide differs from glycogen and starch in terms of glucan chain length.

Amylopectin↗

PubMed2004

Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores.

Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration ([Ca(2+)](ER)) reflects a balance between active uptake by Ca(2+)-ATPases and passive efflux via 'leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca(2+)-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the [Ca(2+)](ER) level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked [Ca(2+)](ER) decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the [Ca(2+)](ER) and could therefore supervise many physiological processes, including gene expression and apoptosis.

Calcium↗

PubMed2004

Bcl-2-dependent modulation of swelling-activated Cl- current and ClC-3 expression in human prostate cancer epithelial cells.

Cell shrinkage is an integral part of apoptosis. However, intimate mechanisms linking apoptotic events to the alterations in cell volume homeostasis remain poorly elucidated. We investigated how overexpression of Bcl-2 oncoprotein, a key antiapoptotic regulator, in lymph node carcinoma of the prostate (LNCaP) prostate cancer epithelial cells interferes with the volume-regulated anion channel (VRAC), a major determinant of regulatory volume decrease. Bcl-2 overexpression resulted in the doubling of VRAC-carried swelling-activated Cl(-) current (I(Cl,swell)) and weakened I(Cl,swell) inhibition by store-operated Ca(2+) channel (SOC)-transported Ca(2+). This also was accompanied by substantial up-regulation of ClC-3 protein, a putative molecular candidate for the role of VRAC. ClC-3-specific antibody suppressed I(Cl,swell) in the wild-type and Bcl-2-overexpressing LNCaP cells. Epidermal growth factor treatment of wild-type LNCaP cells, promoting their proliferation, resulted in the enhancement of endogenous Bcl-2 expression and associated increases in ClC-3 levels and I(Cl,swell) magnitude. We conclude that Bcl-2-induced up-regulation of I(Cl,swell), caused by enhanced expression of ClC-3 and weaker negative control from SOC-transported Ca(2+), would strengthen the ability of the cells to handle proliferative volume increases and thereby promote their survival and diminish their proapoptotic potential.

Apoptosis↗

PubMed2004

Siva-1 and an alternative splice form lacking the death domain, Siva-2, similarly induce apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway.

Siva-1 is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27 and of the glucocorticoid-induced TNFR family-related gene, which are two members of the TNFR family expressed on lymphoid cells. Although Siva-1 expression is up-regulated in multiple pathological processes, little is known about the signaling pathway underlying the Siva-induced apoptosis. In this study, we investigated the mechanism of the proapoptotic activity of Siva-1 and an alternative splice form lacking the death domain of Siva-1, Siva-2, in T lymphocytes in which Siva proteins, CD27, and glucocorticoid-induced TNFR family-related gene are primarily expressed. Overexpression of Siva proteins triggers a typical apoptotic process manifested by cell shrinkage and surface exposure of phosphatidylserine, and confirmed by ultrastructural features. Siva-induced apoptosis is related to the CD27-mediated apoptotic pathway and results in activation of both initiator and effector caspases. This pathway involves a mitochondrial step evidenced by activation of Bid and cytochrome c release, and is modulated by overexpression of Bcl-2 or Bcl-x(L). The determinants for Siva-induced apoptosis are not contained within the death domain found in the central part of Siva-1, but rather in both the N-terminal and C-terminal regions shared by both Siva proteins. The N-terminal region also participates in the translocation of both Siva proteins into the nuclear compartment. These results indicate that Siva-1 and Siva-2 mediate apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway that likely involves both cytoplasmic and nuclear events.

Alternative Splicing↗

PubMed2004

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

Calcium↗

PubMed2004

On the evolution of erythrocyte programmed cell death: apoptosis of Rana esculenta nucleated red blood cells involves cysteine proteinase activation and mitochondrion permeabilization.

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype. Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy and scanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) a caspase-3-like activity; (iv) a mitochondrial membrane potential (Delta Psi m) loss; and (v) a chromatin condensation and fragmentation. Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also prevent Delta Psi m loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight the conservation of the programmed cell death machinery in erythrocytes across kingdom.

Animals↗

PubMed2003

Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells.

The prostate gland is a rich source of alpha1-adrenergic receptors (alpha1-ARs). alpha1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of alpha1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models - primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line - we identify an alpha1A subtype of adrenergic receptor (alpha1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of alpha1A-ARs and DAG analogues activated similar cationic membrane currents and Ca(2+) influx. These currents were sensitive to the alpha1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2-aminophenyl borate and SK&F 96365. Chronic activation of alpha1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by alpha1A-AR and TRP inhibitors. Collectively, our results suggest that alpha1-ARs play a role in promoting hPCE cell proliferation via TRP channels.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2002

Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells.

Antiapoptotic oncoprotein Bcl-2 has extramitochondrial actions due to its localization on the endoplasmic reticulum (ER); however, the specific mechanisms of such actions remain unclear. Here we show that Bcl-2 overexpression in LNCaP prostate cancer epithelial cells results in downregulation of store-operated Ca(2+) current by decreasing the number of functional channels and inhibiting ER Ca(2+) uptake through a reduction in the expression of calreticulin and SERCA2b, two key proteins controlling ER Ca(2+) content. Furthermore, we demonstrate that Ca(2+) store depletion by itself is not sufficient to induce apoptosis in Bcl-2 overexpressing cells, and that sustained Ca(2+) entry via activated store-operated channels (SOCs) is required as well. Our data therefore suggest the pivotal role of SOCs in apoptosis and cancer progression.

Apoptosis↗

PubMed2002

Ligands of the peripheral benzodiazepine receptor are potent inhibitors of Plasmodium falciparum and Toxoplasma gondii in vitro.

The increase in resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands the development of new antimalarial agents. In this quest, we have found that ligands to the peripheral benzodiazepine receptor such as flurazepam, an agonist of the benzodiazepine family, and PK11195, an antagonist derived from isoquinoline, were active against Plasmodium falciparum. These two compounds effectively and rapidly inhibited parasite growth in vitro, irrespective of parasite resistance to chloroquine and mefloquine. Treatment with both drugs induced a sharp and consistent decline in parasitemia, a complete inhibition of parasite replication, and the destruction of parasites within the host red blood cells. Using electron microscopy, we showed that dramatic morphological changes, involving swollen endoplasmic reticulum and the reduction of hemozoin, were consistent with parasite death. The potent activities of flurazepam and PK11195 were also evaluated for antagonist or synergistic effects with currently used antimalarial drugs such as chloroquine and mefloquine. Moreover, flurazepam was found to be active against Toxoplasma gondii, another member of the phylum Apicomplexa. Taken together, our results indicated that benzodiazepines could be considered promising candidates in the treatment of both malaria and toxoplasmosis.

Animals↗

PubMed2002

Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity.

The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and Rz (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.

Bacillus cereus↗

PubMedDate not supplied

Decrease of human hepatoma cell growth by arachidonic acid is associated with an accumulation of derived products from lipid peroxidation.

We showed that the metabolism of arachidonic acid (AA) in HepG2 cells generates reactive oxygen species (ROS), which activate the p38 mitogen-activated protein kinase (MAPK) pathway and the redox-sensitive transcription factors AP-1 and NF-kappaB, leading to the induction of the antioxidant manganese superoxide dismutase gene. The present study reports that AA decreases the HepG2 cell growth by 40% and 55% after a treatment for 24 and 48 h, respectively. This effect was blocked by an inhibitor of lipoxygenase/cytochrome P450 monooxygenase pathways and by the antioxidants. In addition, AA induced an oxidative stress, as an accumulation of malondialdehyde (MDA)-modified proteins, resulting to a generation of MDA and H(2)O(2) was observed after 24 h. This AA-induced oxidative stress was associated with the lack of an increase in the H(2)O(2)-degrading enzyme level. In contrast, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of AA, had not effect. The peroxisome proliferator-activated receptor gamma (PPARgamma) with AA metabolites as ligands was upregulated by the fatty acid but was not involved in the AA effect because its transcriptional activity estimated by reporter gene assays was negatively controlled by p38 MAPK pathway. These findings suggest that the effect of AA on human hepatoma cell growth by inducing an oxidative stress may present a clinical interest in the treatment of the liver cancer.

Arachidonic Acid↗