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The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance [Ca.sup.2+]-activated K ([IK.sub.ca]) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the [IK.sub.Ca] channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or [IK.sub.Ca] channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates [IK.sub.Ca] channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the [IK.sub.Ca] channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce [Ca.sup.2+] mobilization and [IK.sub.Ca] channel activation but only CXCL12 induces a long-term upregulation of the [IK.sub.Ca] channel activity. Furthermore, the [Ca.sup.2+]-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents. chemokines; transwell; tumor; TRAM-34; short hairpin RNA doi: 10.1152/ajpcell.00344.2009.

The effects of histamine on the membrane potential and currents of human glioblastoma (GL-15) cells were investigated. In perforated whole cell configuration, short (3 s) applications of histamine (100 [micro]M) hyperpolarized the membrane by activating a [K.sup.+]-selective current. The response involved the activation of the pyrilamine-sensitive [H.sub.1] receptor and [Ca.sup.2+] release from thapsigargin-sensitive intracellular stores. The histamine-activated current was insensitive to tetraethylammonium (3 mM), iberiotoxin (100 nM), and d-tubocurarine (100 [micro]M) but was markedly inhibited by charybdotoxin (100 nM), clotrimazole (1 [micro]M), and 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34, 1 [micro]M), a pharmacological profile congruent with the intermediate conductance [Ca.sup.2+]-activated [K.sup.+] ([IK.sub.Ca]) channel. Cell-attached recordings confirmed that histamine activated a [K.sup.+] channel with properties congruent with the [IK.sub.Ca] channel (voltage independence, 22 pS unitary conductance and slight inward rectification in symmetrical 140 mM [K.sup.+]). More prolonged histamine applications (2-3 min) often evoked a sustained [IK.sub.Ca] channel activity, which depended on a [La.sup.2+] (10 [micro]M)-sensitive [Ca.sup.2+] influx. Intracellular [Ca.sup.2+] measurements revealed that the sustained [IK.sub.Ca] channel activity enhanced the histamine-induced [Ca.sup.2+] signal, most likely by a hyperpolarization-induced increase in the driving force for [Ca.sup.2+] influx. In virtually all cells examined we also observed the expression of the large conductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.Ca]) channel, with a unitary conductance of ca. 230 pS in symmetrical 140 mM [K.sup.+], and a [Ca.sup.2+] dissociation constant [[K.sub.D(Ca)]] of ca. 3 [micro]M, at -40 mV. Notably in no instance was the [BK.sub.Ca] channel activated by histamine under physiological conditions. The most parsimonious explanation based on the different [K.sub.D(Ca)] for the two [K.sub.Ca] channels is provided. histamine; intermediate conductance [Ca.sup.2+]-activated [K.sup.+] channels; large conductance [Ca.sup.2+]-activated [K.sup.+] channels; human glioblastoma GL-15 cells

Castigli, Emilia, Cataldo Arcuri, Luca Giovagnoli, Roberto Luciani, Leo Giovagnoli, Teresa Secca, Gian Luigi Gianfranceschi, and Virginia Bocchini. Interleukin-1[Beta] induces apoptosis in GL15 glioblastoma-derived human cell line. Am J Physiol Cell Physiol 279: C2043-C2049, 2000.--Interleukin 1-[Beta] (IL-1[Beta]) induces apoptosis in a glioblastoma-derived human cell line, exhibiting a poorly differentiated astrocytic phenotype. The apoptotic effect was demonstrated by analyzing nuclear morphology, in situ DNA fragmentation, and by ELISA detection of cytoplasmatic nucleosomes. We correlated the degree of differentiation of GL15 cells with the apoptotic response: 1) 4', 6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei express low levels of GFAP; and 2) at 13 days of subculture, in a more differentiated state, GL15 cells did not respond with apoptosis to IL-1[Beta]. In this cell line, nonrandom chromosome changes and the expression of SV40 early region have been previously shown. The involvement of p42/p44 mitogen-activated protein kinase (MAPK) pathway in the induction of apoptosis by IL-1[Beta] was hypothesized. Previous studies have shown that SV40 small T antigen partially inhibits phosphatase 2A, leading to an enhancement of the steady-state activity of p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPK pathway, counteracts the apoptotic effect of IL-1[Beta], whereas SB-203580, specific inhibitor of p38 stress-activated protein kinase (SAPK) pathway, is ineffective. The imbalance between MAPK and SAPK pathways has been proposed as a key factor in determination of cell fate. Our results demonstrate that a further stimulation of p42/p44 MAPK pathway can constitute a death signal in tumor cells in which genomic damage and MAPK pathway control alterations occur. p42/p44 mitogen-activated protein kinase pathway

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca2+ -activated K (IKCa) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IKCa channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or IKCa channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced ceil migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IKCa channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IKCa channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca2+ mobilization and IKCa channel activation but only CXCL12 induces a long-term upregulation of the IKCa channel activity. Furthermore, the Ca2+-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents. [ABSTRACT FROM AUTHOR]

We report here the expression and properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IKCa current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent down-regulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ∼35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ∼50% reduction with time in culture. [ABSTRACT FROM AUTHOR]

We have shown that the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) also blocks the intermediate-conductance Ca2+-activated K+ (IKCa) current in human leukemic HL-60 and glioblastoma GL-15 cell lines. The macroscopic IKCa current was activated by ionomycin plus 1-EBIO, and identified as intermediate conductance by being fully blocked by charybdotoxin, clotrimazole, nitrendipine (L-type Ca2+ channel blocker), and NS1619 (BKCa channel opener), but not by d-tubocurarine or TEA. The IKCa current was blocked by NPPB in a reversible dose-dependent manner, with an IC50 of 39 μM in HL-60 and 125 μM in GL-15 cells. The block of the IKCa current was also recorded at the single channel level in excised inside-out patches. As expected, NPPB also blocked the volume-activated Cl− current expressed by GL-15 cells, with an IC50 of 44 μM. The functional implications of IKCa current block by NPPB are discussed. [Copyright &y& Elsevier]

Investigates the effects of interleukin-1 on astrocytoma and glioma cell growth. Effects of interleukin-1 beta on GL15 cells; Effects of interleukin-1 beta on GL15 cell DNA synthesis; Effects of interleukin-1 beta on GL15 apoptosis.