Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 M bradykinin inhibited I _M by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 M and 1 M PDBu diminished I _M to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 M significantly reduced the effect of 1 M PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 M), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on I _M is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of I _M by 1 M PDBu was fully developed, 0.1 M bradykinin produced a further inhibition of I _M. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 M bradykinin significantly but did not abolish it. Staurosporine (0.3 M, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.     

The effect of arachidonic acid on the M current of NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K⁺ current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied the effect of arachidonic acid, other fatty acids and inhibitors of the arachidonic acid metabolism. In relatively high concentrations (25–50 M) arachidonic acid first increased and later decreased the current, I _h, which holds the membrane potential at –30 mV and mainly flows through open M channels. It shifted the midpoint potential, V _o, of the relation between M conductance, g _M, and membrane potential, V, to more negative values and decreased the maximum conductance ¯g _M and the time constant _M. In smaller concentrations (5–10 M) arachidonic acid merely decreased I _h and ¯g _M with little effect on V _o and _M. Eicosatetraynoic acid and docosa-hexaenoic acid acted similarly to arachidonic acid whereas stearic acid had no effect. Of the three enzyme inhibitors studied, nordihydroguaiaretic acid acted similarly to arachidonic acid. i. e. caused a biphasic change in I _h. Indomethacin and quinacrine caused, respectively, a pure increase and a pure decrease of I _h and ¯g _M. Possible explanations are build-up of internally produced arachidonic acid, depletion of eicosanoid products or an inhibitory effect unrelated to arachidonic acid metabolism.     

Activation of nucleotide receptors inhibits M-type K current [I K(M)] in neuroblastoma × glioma hybrid cells

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma × glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K⁺ current [I _K(M) or M-current] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited I _K(M) by 44% and 42%, respectively, at 100 M. Mean IC₅₀ values were: UTP, 0.77±0.27 M; ATP, 1.81±0.82 M. The order of nucleotide and nucleoside activity at 100 M was: UTP = ATP > ATP[S] = ITP > 2 MeSATP > ADP = GTP AMP-CPP, adenosine, where ATP[S] is adenosine 5-O-(3-thiotriphosphate), ITP is inosine 5-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is , methylene ATP. This rank order accords with their activities at the cloned P_2U receptor. Effects were not inhibited by suramin (up to 500 M) or by pre-incubation for 12 h in 500 ng·ml^–1 Pertussis toxin. Inhibition of I_K(M) was frequently preceded by a transient outward current, probably a Ca²⁺-activated K⁺ current, responding to Ca²⁺ mobilization. No effect on the delayed rectifier K⁺ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.Shemyakin Institute of Bio-organic Chemistry, on leave from the Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia     

γδTFH cells promote B cell maturation and antibody production in neuroblastoma

Background

Previous studies have shown that γδ TFH cells are capable of modulating antibody production in immunized and infected mouse model. In recent studies, human γδ TFH cells are shown to contribute to the activation of humoral immunity and promote the maturation of B cells. However, little information is available on their involvement in neuroblastoma (NB) pathogenesis.

Results

In the present study, the frequency of γδ TFH cells in 74 NB patients was significantly higher compared with that in 60 healthy controls. Moreover, most γδ TFH cells in NB patients had a naive phenotype with up-regulation of CD25, CD69, HLA-DR and CD40L and down-regulation of ICOS. Importantly, γδ TFH cells in NB patients produced more IL-4 and IL-10 than those in healthy controls. Furthermore, serum total IgG level was significantly increased in NB patients compared with healthy controls. The expression of CD23 on B cells was up-regulated while CD80 expression was significantly down-regulated in NB patients. Further analysis of B cell compartment showed that the frequency of CD19⁺CD27^hi plasma cells was enhanced in NB patients. Spearman’s correlation analysis revealed that the frequency of γδ TFH cells was positively correlated to serum total IgG level and CD19⁺CD27^hi plasma cells in NB patients, but negatively correlated to CD19⁺ B cells.

Conclusions

We concluded that γδ TFH cells might promote B cell maturation and antibody production in NB patients.

     

Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108–15 cells

 The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca²⁺ channels was investigated in the neuroblastoma × glioma cell line NG108–15 using whole-cell voltage clamp methods. In in vitro differentiated NG108–15 cells, the opioid agonist [d-ala²]-methionine-enkephalin (DAME) reversibly decreased ω-conotoxin-GVIA-sensitive Ba²⁺ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of –40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to –40 mV was a much slower process (τ=88 ms) than the onset of facilitation at +80 mV (τ=7 ms). These results provide evidence that N-type Ca²⁺ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing. Received: 14 September 1998 / Accepted: 29 September 1998     

EGFP–EGF1-conjugated nanoparticles for targeting both neovascular and glioma cells in therapy of brain glioma

As neovascular and glioma cells were closely associated and might be mutually promoted in glioma growth, a dual-targeting strategy targeting to both neovascular and glioma cells would be more promising as compared with those targeting one of them. In this study, we reported a drug delivery system where nanoparticles were decorated with EGFP–EGF1 (ENP), a fusion protein derived from factor VII with special affinity for tissue factor (TF) over-expressed in glioma tissues, to facilitate anti-glioma delivery of paclitaxel (PTX) by targeting both neovascular and glioma cells. In vitro protein binding assay demonstrated that EGFP–EGF1 bound well to C6 cells and perturbed human umbilical vein endothelial cells (HUVEC) with a concentration-dependent manner but not to unperturbed HUVEC. EGFP–EGF1–TF interaction significantly enhanced nanoparticles uptake by perturbed HUVEC and glioma C6 cells as well as nanoparticles penetration in C6 glioma spheroids, and thus improved the cytotoxicity of their payload in both monolayer cells and glioma spheroids models. In vivo imaging of glioma-bearing mice demonstrated the specific accumulation of ENP in glioma tissues. In vivo distribution of nanoparticles intuitively showed ENP mainly sited in both extravascular glioma cells and neovascular cells. Pharmacodynamic results revealed that PTX-loaded ENP (ENP–PTX) significantly prolonged the median survival time of glioma-bearing mice compared with that of any other group. TUNEL assay and H&E staining showed that ENP–PTX treatment induced significantly more cell apoptosis and tumor necrosis compared with other treatments. In conclusion, the results of this contribution demonstrated the great potential of EGFP–EGF1-functionalized nanoparticles for dual-targeting therapy of brain glioma.     

IKBKE is over-expressed in glioma and contributes to resistance of glioma cells to apoptosis via activating NF-κB

IκB kinase-ε (IKBKE), a member of the IκB kinase (IKK) family, has been identified as an oncogenic protein and found to be up-regulated in breast cancer, ovarian cancer and prostate cancer. Nonetheless, the expression status and functional significance of IKBKE in human glioma remain unexplored. For the first time, we have demonstrated that mRNA and protein levels of IKBKE were robustly up-regulated in glioma cell lines and human primary glioma tissues. Immunohistochemistry analysis revealed that 53.5% (38/71) paraffin-embedded archived glioma specimens exhibited high levels of IKBKE expression. Intriguingly, there was no significant difference in IKBKE expression among different grades of glioma. To understand the biological function of IKBKE in the development and progression of human glioma, glioma cells lines ectopically over-expressing IKBKE were established and tested for their responsiveness to apoptotic inducers. Our data showed that IKBKE over-expression inhibited cell apoptosis induced by UV irradiation or adriamycin and, in contrast, shRNAi-mediated suppression of IKBKE increased the sensitivity of glioma cells to the apoptotic inducers. Importantly, we found that up-regulated IKBKE could induce the expression of Bcl-2 through activating NF-κB signalling, and that, specifically, we identified IκB as a critical component for this signalling cascade. The current study suggests that up-regulation of IKBKE may represent an important molecular hallmark that is biologically and clinically relevant to the development and progression, as well as the chemo- and radio-resistance, of the disease.     

Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma×fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes

Intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 M acetylcholine (ACh) increased [Ca²⁺]_i in all four subclones. The increased [Ca²⁺]_i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca²⁺]_i showed sinusoidal oscillations. ACh-induced increases in [Ca²⁺]_i were not observed in cells treated with an intracellular Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA). Removal of extracellular Ca²⁺ with ethyleneglycol-bis-(-aminoethyl)-N,N,N,N'-tetraacetate (EGTA) did not affect the initial [Ca²⁺]_i increases, but reduced the late phases of [Ca²⁺]_i in m1- and m3-transformed cells by 20–30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced [Ca²⁺]_i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50–100 ng ml^–1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1 and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cADPR) produced a rise in [Ca²⁺]_i. ACh-induced [Ca²⁺]_i oscillations were not observed in ryanodine-treated m2-transformed cells. These results show that, while all four mAChRs utilize Ca²⁺ as a common second messenger, m2 and m4 receptors use a different signalling pathway to that used by m1 and m3 receptors.     

Both linopirdine- and WAY123,398-sensitive components of I K(M,ng) are modulated by cyclic ADP ribose in NG108–15 cells

The "M-like" current in NG108-15 cells has two components carried by different K+ channels: a fast-deactivating component, analogous to IK(M) in sympathetic neurones and carried by KCNQ2/3 channels, and a more slowly deactivating component carried by murine erg1 (merg1) channels. The former is selectively blocked by linopirdine (< or =10 microM), the latter by WAY123,398 (< or =10 microM). Bradykinin (100 nM) inhibited 76% of the KCNQ component of current compared with 12% of the merg component. Cyclic ADP ribose (cADPR, 2 microM), introduced via the patch pipette, caused a rundown of both current components. Acetylcholine (100 microM) inhibited 89% of the KCNQ component of current compared to 34% of the merg component. After 15 min of intracellular dialysis with the cADPR antagonist 8-amino-cADP ribose (100 microM), the inhibition reduced to 40% and 19% and after 30 min it was further reduced to 8% and 5% for the KCNQ currents and merg currents respectively. These data show that both KCNQ and merg currents in NG108-15 cells can be modulated by either bradykinin or M1 muscarinic receptors. The inhibition of the KCNQ current component is more pronounced than that of the merg component. These results suggest that cADPR might be involved in M1-muscarinic inhibition of both KCNQ2/3 and merg1 channels.     

Correlations between alterations in length-dependent Ca2+ activation of cardiac myofilaments and the end-systolic pressure–volume relation

We have tested the hypothesis that alterations in length dependent activation (LDA) of cardiac myofilaments represent an important regulatory mechanism affecting the Frank-Starling mechanism as determined by the slope (E(es)) of the relation between left ventricular (LV) volume and end-systolic pressure. We employed a transgenic (TG) mouse model in which the cardiac isoform of TnI (cTnI) has been completely replaced with slow skeletal TnI (ssTnI), the embryonic/neonatal isoform in the heart. Compared to non-transgenic (NTG) controls, myofilaments from TG-ssTnI hearts demonstrate an increase in Ca(2+) sensitivity and a substantially blunted LDA that is unaffected by PKA-dependent phosphorylation. We measured in situ LV pressure and volume relations during basal conditions and isoproterenol (ISO) stimulation. In the basal state in TG-ssTnI hearts there was significant increase in end-systolic pressure and slight decrease in heart rate. ISO stimulation resulted in a significant increase in heart rate, ejection fraction, maximum dP/dt, preload-recruitable stroke work, maximum dP/dt versus end diastolic volume and cardiac output in both groups. During basal conditions there was no difference in the E(es) relation between NTG and TG-ssTnI groups. However, during ISO stimulation the E(es) relation was significantly different between NTG and TG-ssTnI groups. Our study provides the first direct evidence that enhancement in differences in LDA between cardiac myofilaments from NTG and TG-ssTnI hearts induced by post-translational modifications of sarcomeric proteins are reflected in the in situ beating heart by a different change in E(es). Thus, changes in LDA should be considered in interpreting results from in situ experiments on inotropic effects associated with physiological and patho-physiological states of the heart.     

Selectivity of a translation-inhibitory factor,CpBV15β, in host mRNAs and subsequent alterations in host development and immunity

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella. Its symbiotic virus, C. plutellae bracovirus (CpBV), has been shown to play a crucial role in inducing physiological changes in the parasitized host. A viral gene, CpBV15β, exhibits a specific translational control against host mRNAs by sequestering a eukaryotic translation initiation factor, eIF4A. Inhibitory target mRNAs have high thermal stability (>≈9 kcal/mol) of their secondary structures in 5′UTR. To determine the specificity of translational control in terms of 5′UTR complexity, this study screened target/nontarget mRNAs of CpBV15β using a proteomics approach through an in vivo transient expression technique. A proteomics analysis of host plasma proteins showed that 12.9% (23/178) spots disappeared along with the expression of CpBV15β. A total of ten spots were chosen, in which five spots (‘target’) were disappeared by expression of CpBV15β and the other five (‘nontarget’) were insensitive to expression of CpBV15β, and further analyzed by a tandem mass spectroscopy. The predicted genes of target spots had much greater complexity (−12.3 to −25.2 kcal/mol) of their 5′UTR in terms of thermal stability compared to those (−3.70 to −9.00 kcal/mol) of nontarget spots. 5′UTRs of one target gene (arginine kinase:Px-AK) and one nontarget gene (imaginal disc growth factor:Px-IDGF) were cloned and used for in vitro translation (IVT) assay using rabbit reticulocyte lysate. IVT assay clearly showed that mRNA of Px-IDGF was translated in the presence of CpBV15β, but mRNA of Px-AK was not. Physiological significance of these two genes was compared in immune and development processes of P. xylostella by specific RNA interference (RNAi). Under these RNAi conditions, suppression of Px-AK exhibited much more significant adverse effects on larval immunity and larva-to-pupa metamorphosis compared to the effect of suppression of Px-IDGF. These results support the hypothesis that 5′UTR complexity is a molecular motif to discriminate host mRNAs by CpBV15β for its host translational control and suggest that this discrimination would be required for altering host physiology to accomplish a successful parasitism of the wasp host, C. plutellae.     

Inhibition of voltage-dependent Ca2+ channels via α2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamindes and opioids. The effects of these agents on whole-cell Ca²⁺ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca²⁺ channel currents recorded during voltageclamp pulses from a holding potential of –80 mV to 0 mV were reversibly reduced by 10 M epinephrine (in the presence of 1 M propranolol) or 5 M of the synthetic opioid, d-Ala²-d-Leu⁵-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca²⁺ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 M) reduced the Ca²⁺ channel current by approximately 40%; the Ca²⁺ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including G_i1, G_i2, G_o1 and another G_o subtype, possibly G_o2. The data show that activation of ₂-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca²⁺ channels in adrenal chromaffm cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.     

Modulation of Kir4.1 and Kir4.1–Kir5.1 channels by small changes in cell volume

The K⁺ channels Kir4.1 and Kir4.1–Kir5.1 are expressed in the glial cells of the CNS and are involved in regulation of the K⁺ homeostasis. Several studies have shown that Kir4.1 channels are co-localized with aquaporins (AQP4) in the glial endfeet, and a putative functional coupling between the Kir channels and aquaporins is therefore debated. To test a possible volume-sensitivity of the Kir channels, the Kir4.1 or Kir4.1–Kir5.1 channels were expressed in Xenopus oocytes with or without co-expression of aquaporins and subsequently exposed to cell volume alterations. Our results show an increase in Kir4.1 and Kir4.1–Kir5.1 currents upon swelling of the oocytes and a reduction in the current when the oocytes were shrunk. The volume-dependent changes in channel activity were not due to changes in the kinetics of the channels. These findings implicate a putative functional interaction between the Kir channels and aquaporins via small, fast cell volume changes in the glial cells.     

Induction of suppressive phenotype in monocyte-derived dendritic cells by leukemic cell products and IL-1β

Professional antigen-presenting cells, dendritic cells (DCs) play an important role in controlling tumors. It is known that solid tumor cell products inhibit DC differentiation. Recently a similar effect produced by leukemic cell products has been demonstrated. In this case, leukemic cell products induced the secretion of IL-1β by monocytes undergoing differentiation. The aim of the present work was to characterize and to compare the development of monocyte-derived DCs under the influence of leukemic cell products (K562 supernatant) or exogenous IL-1β. It became clear that leukemic cell products and IL-1β differentially modulate some of the parameters studied on monocytes stimulated to differentiate into DCs. In the presence of K562 supernatant, the expression of the macrophage markers CD16 and CD68 were higher than in immature DCs control. Contrasting with IL-1β, leukemic cell products possibly favor the development of cells with macrophage markers. In addition, CD80 and CD83 expressions were also higher in the presence of tumor supernatant whereas HLA-DR was lower. In the presence of IL-1β, only CD80 was increased. Furthermore, it was observed that when monocytes were induced to differentiate into DCs in the presence of tumor supernatant and then activated, they expressed less CD80 and CD83 than activated DCs control. A reduced expression of CD83 following activation was also seen in cells differentiated with IL-1β. TGF-β and VEGF were found in the tumor supernatants. Moreover, the exposure to tumor supernatant or IL-1β stimulated IL-10 production while decreased IL-12 production by activated DCs. Finally, these results suggest that the addition of products released by leukemic cells or, more discreetly, the addition of IL-1β affects DC differentiation, inducing a suppressive phenotype.     

Herpes simplex virus type I induces the accumulation of intracellular β-amyloid in autophagic compartments and the inhibition of the non-amyloidogenic pathway in human neuroblastoma cells

Mounting evidence suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer's disease (AD). Epidemiological analyses have shown that HSV-1 is a risk factor for AD in people with at least 1 type 4 allele of the apolipoprotein E gene. Recent studies have also suggested that HSV-1 contributes to the appearance of the biochemical anomalies characteristic of AD brains. In addition, autophagic activity appears to be reduced with aging, and the final stages of autophagy in neurodegenerative process appear to be impaired. The present work reports that HSV-1 provokes the strong intracellular accumulation of both the main species of β-amyloid (Aβ) in the autophagic compartments and that it is associated with a marked inhibition of Aβ secretion. Autophagosomes containing Aβ failed to fuse with lysosomes in HSV-1-infected cells, indicating the impaired degradation of Aβ localized in the autophagic vesicles. In addition, HSV-1 infection was associated with the inhibition of the nonamyloidogenic pathway of amyloid precursor protein (APP) processing without significantly affecting the activity of the secretases involved in the amyloidogenic pathway. Taken together, these data suggest that HSV-1 infection modulates autophagy and amyloid precursor protein processing, contributing to the accumulation of Aβ characteristic of AD.