Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner.

BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS: Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION: We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS: The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

The disease modifying osteoarthritis drug diacerein is able to antagonize pro inflammatory state of chondrocytes under mild mechanical stimuli.

OBJECTIVE: To investigate the combination of mild mechanical stimuli and a disease modifying osteoarthritis drug (DMOAD) in inflammatory activated chondrocytes and to study the combination of drug and mechanical tension on the cellular level as a model for an integrated biophysical approach for osteoarthritis (OA) treatments. METHODS: Interleukin-1beta (IL-1ß) stimulated C28/I2 cells underwent mild mechanically treatment while cultured in the presence of the DMOAD diacerein. The pharmacological input of diacerein was evaluated by cell viability and cell proliferation measurements. Inflammation and treatment induced changes in key regulatory proteins and components of the extracellular matrix (ECM) were characterized by quantitative real-time PCR (qPCR). The effects on metalloproteinase-1 (MMP-1) activity and glycosaminoglycan (GAG) concentration in cell supernatants of treated cells were investigated. RESULTS: C28/I2 cells demonstrated significant changes in expression of inflammatory and cartilage destructive proteins in response to IL-1ß stimulation. The chondroprotective action of diacerein in mechanically stimulated cells was mediated by a decrease in interleukin-8 (IL-8), fibronectin-1 (FN-1), collagen type I (Col 1) and MMP-1 expression levels, respectively. Augmented expression of interleukin-6 receptor (IL-6R) and the fibroblast growth factor receptors (FGFRs) by diacerein was not abolished by mechanical treatment. The observed effects were accompanied by a reduced cell proliferation rate, attenuated cell viability and extenuated MMP-1 activity. CONCLUSION: Diacerein diversely regulates the expression of main regulatory proteins as well as components important to regenerate and set up ECM. Mechanical stimulation does not negatively influence the chondroprotective effect induced by diacerein treatment in immortalized human C28/I2 chondrocytes.

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated alpha-tocopheryl-succinate.

Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.

Quantification of apolipoprotein D by an immunoassay with time-resolved fluorescence spectroscopy.

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6 +/- 32 microg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6 +/- 1.4 and 2.7 +/- 0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.

Modulation of the human cardiac sodium channel alpha-subunit by cAMP-dependent protein kinase and the responsible sequence domain.

1. In order to investigate the modulation of human hH1 sodium channel alpha-subunits by cAMP-dependent protein kinase (PKA), the channel was expressed in oocytes of Xenopus laevis. 2. Cytosolic injection of cAMP, as well as of SP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (SP-cAMPS, the S-diastereoisomeric configuration of the compound with respect to the phosphorus atom), resulted in a marked and significant increase in peak sodium current (INa,p). Cytosolic injections of RP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (RP-cAMPS; a compound inhibitory to PKA) had no effect on peak current. 3. Kinetic parameters of steady-state activation, inactivation and recovery from inactivation were unchanged following stimulation of PKA activity, but a 42 +/- 5% (mean +/- S.E.M.) increase in maximal sodium conductance (delta gmax) could account for the observed increase in INa,p. 4. A set of chimerical sodium channels made from portions of the human cardiac hH1 alpha-subunit and the rat skeletal muscle SkM1 alpha-subunit (which is not affected by PKA stimulation) was generated. These were used to localize the structural determinant in the hH1 sequence responsible for PKA modulation of hH1. From our data we conclude that the effects of PKA on hH1 are conferred by the large cytosolic loop interconnecting transmembrane domains I and II, which is not conserved among sodium channel subtypes.

Modulation of cardiac sodium channel isoform by cyclic AMP dependent protein kinase does not depend on phosphorylation of serine 1504 in the cytosolic loop interconnecting transmembrane domains III and IV.

Both the neuronal IIA as well as the cardiac SkM2 isoform of the pore forming alpha-subunit of voltage dependent sodium channels are modulated by Protein Kinase A. While alphaIIA becomes attenuated upon PKA stimulation, alphaSkM2 becomes upregulated. PKC dependent phosphorylation of a serine, located in the highly conserved cytoplasmatic region between the third and the fourth transmembraneous domain has been found to be a prerequisite for PKA modulation of the alphaIIA isoform. We used site-directed mutagenesis, expression in Xenopus laevis oocytes and the two-electrode voltage clamp technique to test, whether phosphorylation of the corresponding serine in alphaSkM2 is required for the PKA modulation of also the cardiac isoform. The results clearly indicate that serine 1504 does not play a significant role in the PKA modulation of the cardiac sodium channel isoform, further underlining the differential modulation of the two isoforms by identical signal transduction cascades.

Beta-adrenergic modulation of currents produced by rat cardiac Na+ channels expressed in Xenopus laevis oocytes.

In Xenopus oocytes coexpressing beta 2-adrenergic receptors and the rat cardiac alpha SkM2 Na+ channel, superfusion with 10 microM isoproterenol led to modest (approximately 30%) increases in peak Na+ inward current. Intracellular injection of cAMP and of protein kinase A (PKA) catalytic subunit reproduced this increase, showing that the second messenger pathway involves PKA dependent phosphorylation. Coexpression of the Na+ channel beta 1 subunit had no influence on the modulation. The modulation had little or no effect upon Na+ current waveforms, steady-state activation, steady-state activation, steady-state inactivation, or recovery from both fast and slow inactivation; but maximum Na+ conductance was increased. Mutation of the five major consensus PKA phosphorylation sites on alpha SkM2 did not abolish the observed effect. In parallel experiments, beta-adrenergic stimulation of the neuronal alpha IIA Na+ channel subunit led to an attenuation of Na+ current. It is concluded that (i) the alpha SkM2 subunit might be directly phosphorylated by PKA, but at serine/threonine residue(s) in a cryptic phosphorylation site(s); or that (ii) the modulation might also be mediated by phosphorylation of another, as yet unknown protein(s). The divergent modulation of neuronal and cardiac Na+ channel alpha-subunits suggests that differential physiological modulation by identical second messenger pathways is the evolutionary basis for the isoform diversity within this protein family.

Factors influencing the phagocytosis-stimulated glucose oxidation in porcine leukocytes.

The stimulation of the D-(1-14C)glucose and D-(6-14C)glucose metabolism in pig leucocytes during the phagocytosis of bacteria and inert particles was studied. The following results were obtained: 1. The magnitude of phagocytosis-stimulated glucose oxidation is directly related to the nature and number of particles added. 2. Live bacteria stimulate the glucose metabolism to a greater extent than do a similar number of heat-killed organisms. As to the extent of the stimulation the species of bacteria offered for phagocytosis is crucial. 3. After in vitro addition of a lipopolysaccharide a stimulating effect is observed, a depression has been shown after the addition of hydrocortisone.