CXCR7-mediated progression of osteosarcoma in the lungs.

BACKGROUND: Osteosarcoma (OS) is the most frequent primary malignant bone tumour in children and adolescents with a high propensity for lung metastasis. Chemokines and chemokine receptors have been described to have an important role in many malignancies including OS. The aim of this study was to investigate the expression of CXCR7 receptor in OS tissues and its role in the progression of the disease in the lungs. METHODS: Immunohistochemistry was used to study CXCR7 expression in primary tumours and metastatic tissues from patients with OS. Its contribution to tumour expansion in the lungs has been also assessed using animal models and synthetic-specific CXCR7 ligands. RESULTS: CXCR7 was expressed on human primary bone tumours and on lung metastases. Its expression was predominantly located on tumour-associated blood vessels. Mice challenged with OS cells and systematically treated with synthetic CXCR7 ligands presented a significant reduction of lung nodules compared with untreated mice. CONCLUSION: This study shows that CXCR7 has a critical role in OS progression in the lungs, where are expressed CXCR7 ligands, especially CXCL12. Moreover, we highlight that synthetic CXCR7 ligands could represent a powerful therapeutic tool to impede lung OS progression.

CXCR7 receptors facilitate the progression of colon carcinoma within lung not within liver.

BACKGROUND: Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Chemokine-receptor pairs have a critical role in determining the metastatic progression of tumours. Our hypothesis was that disruption of CXCR7/CXCR7 ligands axis could lead to a decrease in CRC metastases. METHODS: Primary tumours and metastatic tissues from patients with CRC were tested for the expression of CXCR7 and its ligands. Relevance of CXCR7/CXCR7 ligands for CRC metastasis was then investigated in mice using small pharmacological CXCR7 antagonists and CRC cell lines of human and murine origins, which - injected into mice - enable the development of lung and liver metastases. RESULTS: Following injection of CRC cells, mice treated daily with CXCR7 antagonists exhibited a significant reduction in lung metastases. However, CXCR7 antagonists failed to reduce the extent of liver metastasis. Moreover, there were subtle differences in the expression of CXCR7 and its ligands between lung and liver metastases. CONCLUSION: Our study suggests that the activation of CXCR7 on tumour blood vessels by its ligands may facilitate the progression of CRC within lung but not within liver. Moreover, we provide evidence that targeting the CXCR7 axis may be beneficial to limit metastasis from colon cancer within the lungs.

Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism.

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.

Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels.

Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2.

Recently c-Cbl has been reported to be phosphorylated upon CSF-1 stimulation. The product of the c-cbl proto-oncogene (c-Cbl) is a 120 kDa protein harboring several docking sites for Src homology 2 (SH2) domain containing proteins and proline-rich regions that have been shown to allow its constitutive association with the SH3 domains of Grb2. We demonstrate here that CSF-1 exposure of stable transfectant CHO cells expressing the CSF-1 receptor induced the sustained tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 kDa subunit of the PI 3-kinase, while it constitutively associates with Grb2. We demonstrate by in vitro experiments that these associations require the SH2 domain of Crk-II and both the C- and N-terminal SH2 domains of the p85 subunit of the PI 3-kinase. cCbl is the major PI 3-kinase-containing protein in c-Fms expressing CHO cells upon CSF-1 stimulation. Thus c-Cbl behaves as a core protein, allowing the formation of a quaternary complex including, Crk-II, PI 3-kinase and Grb2. We provide evidence that this multiprotein complex can interact with the tyrosine phosphorylated CSF-1 receptor through the unoccupied SH2 domain of Grb2.

Platelet-activating factor activates a Ca(2+)-dependent K+ channel which is not involved in c-fos expression in human B lymphoblastoid cells.

In this report, it is shown that the platelet-activating factor (PAF) induced, in human B lymphoblastoid cells, 86Rb+ influx and efflux suggesting that it activated a K+ channel. Opening of this channel was dependent on PAF-induced Ca2+ mobilization. Ionomycin and thapsigargin--a specific inhibitor of (Ca(2+)-Mg2+)-ATPase--mimicked the effect of PAF both on intracellular calcium and activation of the channel. This channel was inhibited by charybdotoxin, high doses of tetraethylammonium and barium but was insensitive to apamin, 4-aminopyridine. These features indicate that PAF activated a Ca(2+)-dependent K+ channel. In these cells, PAF also induced the expression of c-fos oncogene. This effect was not affected by charybdotoxin indicating that this channel is not involved in the control of early gene transcription.

Somatostatin activates glibenclamide-sensitive and ATP-regulated K+ channels in insulinoma cells via a G-protein.

Somatostatin, an hyperglycemia-inducing hormone, was studied in rat insulinoma (RINm5F) cells using 86Rb+ efflux techniques. 86Rb+ efflux is stimulated by somatostatin in a dose-dependent manner. The half-maximum value of activation is 0.7 nM. Somatostatin-induced 86Rb+ efflux is abolished by the hypoglycemia-inducing sulfonylurea, glibenclamide, a known blocker of ATP-regulated K+ channels. Somatostatin activation is prevented by pretreatment of insulinoma cells with pertussis toxin. 86Rb+ efflux studies show that somatostatin activates an ATP-dependent K+ channel.

The apamin-sensitive Ca2+-dependent K+ channel molecular properties, differentiation and endogenous ligands in mammalian brain.

Apamin is a bee venom neurotoxin of 10 amino acids containing two disulphide bridges. Current-clamp and voltage-clamp experiments have shown that apamin externally applied blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylamonium-insensitive. It is distinct from the high conductance Ca+-dependent K+ channel revealed by patch-clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low: between 1 and 40 fmol/mg of protein. Radiation-inactivation analysis indicates a molecular mass for the apamin receptor of 250 000 Da whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular mass of about 30 000 Da. Autoradiography of 125I-apamin binding sites reveals the presence of Ca2+-activated K+ channels in many regions of the brain. There is an all-or-none control of the expression of the apamin-sensitive Ca2+-dependent K+ channel by innervation in mammalian skeletal muscle. There exists an endogenous equivalent of apamin in rat brain.

86Rb+ transport in Leishmania infantum promastigotes under various in vitro culture conditions.

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on 86Rb+ influx in L. infantum promastigotes. Proliferating promastigotes exhibited faster and higher 86Rb+ uptake than stationary cells. Cl- anion did not have any effect, but in the presence of physiological concentration of HCO3-, 86Rb+ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. 86Rb+ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased 86Rb+ influx. Our data provide indirect evidence that an energy-dependent system which brings K+ in, such as K+/H(+)-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPases occur, at least in the presence of HCO3-, and their contribution to K+ pathways varies in different environmental conditions.

Galanin inhibits dopamine secretion and activates a potassium channel in pheochromocytoma cells.

Galanin inhibits depolarization-induced dopamine release from chromaffin cells. In excised membrane patches, galanin induced openings of a 36 pS, inwardly rectifying potassium channel. Galanin activation of this K+ channel was blocked by pretreatment with pertussis toxin. Galanin is without effect on L-type Ca2+ channels.

Arg777 plays a major role in the conformation of the colony-stimulating factor-1 receptor intracellular kinase domain.

A point mutation substituting Arg777 by Gln was obtained in a highly conserved region of the human colony-stimulating factor-1 receptor (CSF-1R) sequence. Constitutive expression of wild-type receptors in CHO cells confers susceptibility to CSF-1 for proliferation whereas the mutated receptors exhibited a 90% reduced efficiency in proliferation. We sought to determine the alterations intervening in the CSF-1 signal transduction of the Arg777Gln mutated receptor. We found that ligand binding and ligand-induced CSF-1R internalization were unaffected. CSF-1-induced receptor dimerization and autophosphorylation were impaired to the same extent as mitogen-activated protein kinase activation (90%). However, only phosphatidylinositol 3-kinase activation and ligand-induced receptor ubiquitination were abrogated by the mutation. These features probably reflect the inability of the mutated CSF-1R kinase domain to fold properly and hence to autophosphorylate and/or to associate correctly with transduction proteins. These data may indicate a role for the conserved regions of the RTK kinase domains in the stabilization of the intracellular domain conformation.

Effect of fractalkine-Fc delivery in experimental lung metastasis using DNA/704 nanospheres.

The lung is one target organ to which solid tumors frequently metastasize. Given the systemic adverse effects of currently available treatments, developing effective strategies of drug/gene delivery directly to the lungs is therefore needed. Aerosol delivery is a non-invasive gene transfer approach to target the airways. Here, we sought to evaluate the potential to deliver a fractalkine (FKN)-encoding plasmid formulated with the tetrafunctional amphiphilic block copolymer 704 through aerosolization in two models of pulmonary metastases. FKN is a chemokine recently described as a good candidate to stimulate a strong antitumor immune response in various forms of cancers. Here, we have assessed the effect of single and repeated aerosolizations of FKN-encoding plasmid formulated with 704 on the development of experimental lung metastases of mouse colon carcinoma and osteosarcoma. For this purpose, we have designed FKN-Fc sequences encoding an optimized version of the chemokine. Repeated intratracheal administrations of 704/FKN-Fc markedly inhibited growth of experimental lung metastases of CT-26 and K7M2 cells. Our results showed that tetrafunctional amphiphilic block copolymer 704 is a highly efficient synthetic vector for mediating local and safe gene transfer into the lung. In addition, FKN-Fc gene therapy of pulmonary nodules may provide a promising immunotherapeutic approach.

Tissue-specific differential antitumour effect of molecular forms of fractalkine in a mouse model of metastatic colon cancer.

BACKGROUND AND AIMS: Fractalkine, a chemokine that presents as both a secreted and a membrane-anchored form, has been described as having tumour-suppressive activities in standard subcutaneous models. Here, we investigate the antitumour effect of fractalkine, in its three molecular forms, in two orthotopic models of metastatic colon cancer (liver and lung) and in the standard subcutaneous model. METHODS: We have developed models of skin tumours, liver and pulmonary metastasis and compared the extent of tumour development between C26 colon cancer cells expressing either the native, the soluble, the membrane-bound fractalkine or none. RESULTS: The native fractalkine exhibits the strongest antitumour effect, reducing the tumour size by 93% in the skin and by 99% in the orthotopic models (p<0.0001). Its overall effect results from a critical balance between the activity of the secreted and the membrane-bound forms, balance that is itself dependent on the target tissue. In the skin, both molecular variants reduce tumour development by 66% (p<0.01). In contrast, the liver and lung metastases are only significantly reduced by the soluble form (by 96%, p<0.002) whereas the membrane-bound variant exerts a barely significant effect in the liver (p = 0.049) and promotes tumour growth in the lungs. Moreover, we show a significant difference in the contribution of the infiltrating leukocytes to the tumour-suppressive activity of fractalkine between the standard and the orthotopic models. CONCLUSIONS: Fractalkine expression by C26 tumour cells drastically reduces their metastatic potential in the two physiological target organs. Both molecular forms contribute to its antitumour potential but exhibit differential effects on tumour development depending on the target tissue.

Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor.

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.

Apamin: a specific toxin to study a class of Ca2+-dependent K+ channels.

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.

Antidiabetic sulfonylureas control action potential properties in heart cells via high affinity receptors that are linked to ATP-dependent K+ channels.

Both avian and mammalian heart cells have high affinity receptors for antidiabetic sulfonylureas. The biochemical identification of these receptors has been carried out with [3H]glibenclamide. The Kd values for the most potent sulfonylureas, such as glibenclamide itself, are in the nanomolar range. Comparative studies of structure-function relationships indicate high similarities of binding properties between the sulfonylurea receptors in cardiac cells and insulinoma cells, respectively. The duration of the action potential of guinea pig cardiac cells was drastically reduced by decreasing intracellular ATP concentrations by perfusion or by blockade of oxidative phosphorylation. Glibenclamide was found to restore normal or nearly normal action potential properties in [ATP]in-depleted cardiac cells. Single channel recording using the patch-clamp technique has shown that this effect is associated with high affinity blockade of ATP-sensitive K+ channels by sulfonylureas.