HOE-BAY 946 has distinct effects on spontaneous and FGF induced proliferation of epithelial and neuroblastoma cell lines (original) (raw)

Protein kinase C isoforms and cell proliferation in neuroblastoma cells

Molecular Brain Research, 1996

The expression of protein kinase C isoforms in the neuroblastoma cell line Neuro 2a has been studied. It is shown that Neuro 2a cells express c~, 8, E and ,~ PKCs. Inhibition of cell proliferation by using protein kinase C inhibitors (H7 or calphostin C) or medium without glutamine affects markedly the pattern of PKC isoforms. All treatments reduced significantly (50-70%) the content of PKC a. None of the treatments altered PKC ~" or E. The content of PKC 8 was increased (88-120%) in cells treated with PKC inhibitors but was slightly reduced in cells incubated in medium without glutamine. However, none of the treatments affected the content of the corresponding mRNAs. Long-term treatment of synchronized cells with the phorbol ester PMA depletes PKC a but not PKC 6 or ~" and only partially PKC e. This treatment with PMA did not affect DNA synthesis, indicating that PKC c~ does not play a significant role in the control of proliferation of these cells.

FGF1 C-terminal domain and phosphorylation regulate intracrine FGF1 signaling for its neurotrophic and anti-apoptotic activities

Cell death & disease, 2016

Fibroblast growth factor 1 (FGF1) is a prototypic member of the FGFs family overexpressed in various tumors. Contrarily to most FGFs, FGF1 lacks a secretion peptide signal and acts mainly in an intracellular and nuclear manner. Intracellular FGF1 induces cell proliferation, differentiation and survival. We previously showed that intracellular FGF1 induces neuronal differentiation and inhibits both p53- and serum-free-medium-induced apoptosis in PC12 cells. FGF1 nuclear localization is required for these intracellular activities, suggesting that FGF1 regulates p53-dependent apoptosis and neuronal differentiation by new nuclear pathways. To better characterize intracellular FGF1 pathways, we studied the effect of three mutations localized in the C-terminal domain of FGF1 (i.e., FGF1(K132E), FGF1(S130A) and FGF1(S130D)) on FGF1 neurotrophic and anti-apoptotic activities in PC12 cells. The change of the serine 130 to alanine precludes FGF1 phosphorylation, while its mutation to aspartic...

Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells

Biochemical and Biophysical Research Communications, 2003

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 lM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factorinduced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.

H7, an inhibitor of protein kinase C, prevents serum-induced phosphorylation of Raf and MAP kinase in neuroblastoma cells

Neuroscience Letters, 1996

We have previously shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, inhibits proliferation of neuroblastoma cells in culture. We have now tested whether the effect of H7 is mediated by MAP kinase and Raf. It is shown that, in Neuro 2a cells, activation of protein kinase C by addition of 4~3-phorbol-12/3-myristate-13~-acetate (PMA), leads to phosphorylation of Raf and Mitogen-activated protein kinase (MAP kinase). PMA-induced phosphorylation of these proteins is prevented by H7. When quiescent Neuro 2a were stimulated to proliferate by addition of serum, Raf and MAP kinase were rapidly phosphorylated. Serum-induced phosphorylation of Raf and MAP kinase is prevented by H7. These results suggest that, in Neuro 2a cells, the control of proliferation by protein kinase C could be mediated by phosphorylation (and concomitant activation) of Raf and MAP kinase.

Protein kinase inhibitor, staurosporine, induces a mature neuronal phenotype in SH-SY5Y human neuroblastoma cells through an ?-, ?-, and ?-protein kinase C-independent pathway

Journal of Cellular Physiology, 1993

Previous studies have shown that the tumour-promoting phorbol ester 12-0tetradecanoyl phorbol-13 acetate (TPA) induces both morphological and functional differentiation in SH-SY5Y human neuroblastorna cells (Pshlrnan et al., 1981). In order to investigate the role of protein kinase C (PKC) in TPA-induced maturation of SH-SY5Y cells, we have used staurosporine, which is a potent inhibitor of protein kinases including PKC. Treatment of SH-SY5Y cells with 25 n M staurosporine for 72 hours caused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was accompanied by a cessation of DNA synthesis, induction of growth associated protein 43 (GAP-43), and neuropeptide Y (NPY) rnRNA. These effects of staurosporine were comparable to those elicited by TPA. Staurosporine further induced a time-dependent increase in the expression of tyrosine hydroxylase protein and a SO-fold increase in the concentration of noradrenaline. TPA only induced a marginal increase in tyrosine hydroxylase expression. Both TPA and staurosporine induced an appearance of voltage-gated Ca'+ channels in SH-SY5Y cells detected with single-cell fluorescent measurements using fura-2. The Ca'+ channels were found almost exclusively in growth cones and varicosities. Staurosporine inhibited both basal and a TPA-induced phosphorylation of an endogenous 80kDa PKC substrate (p80), and also blocked c-fos proto-oncogene rnRNA expression induced by the phorbol ester. Bryostatin 1, a potent activator of PKC, has failed to induce morphological or functional differentiation in SH-SY5Y cells (Jalava et al., 1990). Incubation of SH-SY5Y cells in the presence of 100 n M bryostatin 1 for 24 hours caused a complete disappearance of all irnrnunoreactive a-, p-, and 5-PKC. The level of E-PKC decreased by 70%. Staurosporine induced a partial translocation of the &-isoenzyme but it failed to cause down-regulation of E-PKC. Bryostatin 1-treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation of DNA synthesis, and GAP-43 and NPY mRNA expression. The ability of staurosporine to stimulate tyrosine hydroxylase expression and to increase cellular content of noradrenaline was also unaffected. Taken together the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH-SY5Y cells through an a-, p-, and 5-PKC-independent pathway. o 1993 WiIey-Liss, Inc. SH-SY5Y human neuroblastoma cells are induced t o differentiate t o m a t u r e n e u r o n a l cells w h e n treated with tumour-promoting p h o r b o l esters such as TPA (for a review, see PBhlman e t al., 1990). Characteristic t o TPA-treated S H-S Y 5 Y cells i s an appearance o f long, n e u r i t e l i k e processes with varicosities and g r o w t h cones (PBhlman e t al., 1981), an increased expression o f mRNA for axon-specific g r o w t h associated p r o t e i n (GAP-43) (Bjelfman e t al., 1990), elevated concentrations o f noradrenaline and neuron-specific enolase (Pbhlman e t al., 1981), and an appearance of voltage gated Ca2+ channels (H e i k k i l a e t al., 1989). S H-S Y 5 Y 0 1993 WILEY-LISS, INC cell differentiation i s also associated with a down-regul a t i o n o f c-myc proto-oncogene mRNA (H a m m e r l i n g e t al., 1987; Jalava et al., 1988) and p r o t e i n expression (Jalava e t al., 1992) as w e l l as an increased pp6OC-"'" kinase a c t i v i t y (Bjelfman e t al., 1990).

FGF5 as an oncogenic factor in human glioblastoma multiforme: autocrine and paracrine activities

Oncogene, 2008

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 down-modulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/ or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.

Protein kinase inhibitor, staurosporine, induces a mature neuronal phenotype in SH-SY5Y human neuroblastoma cells through an α-, β-, and ζ-protein kinase C-independent pathway

Journal of Cellular Physiology, 1993

Previous studies have shown that the tumour-promoting phorbol ester 12-0tetradecanoyl phorbol-13 acetate (TPA) induces both morphological and functional differentiation in SH-SY5Y human neuroblastorna cells (Pshlrnan et al., 1981). In order to investigate the role of protein kinase C (PKC) in TPA-induced maturation of SH-SY5Y cells, we have used staurosporine, which is a potent inhibitor of protein kinases including PKC. Treatment of SH-SY5Y cells with 25 n M staurosporine for 72 hours caused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was accompanied by a cessation of DNA synthesis, induction of growth associated protein 43 (GAP-43), and neuropeptide Y (NPY) rnRNA. These effects of staurosporine were comparable to those elicited by TPA. Staurosporine further induced a time-dependent increase in the expression of tyrosine hydroxylase protein and a SO-fold increase in the concentration of noradrenaline. TPA only induced a marginal increase in tyrosine hydroxylase expression. Both TPA and staurosporine induced an appearance of voltage-gated Ca'+ channels in SH-SY5Y cells detected with single-cell fluorescent measurements using fura-2. The Ca'+ channels were found almost exclusively in growth cones and varicosities. Staurosporine inhibited both basal and a TPA-induced phosphorylation of an endogenous 80kDa PKC substrate (p80), and also blocked c-fos proto-oncogene rnRNA expression induced by the phorbol ester. Bryostatin 1, a potent activator of PKC, has failed to induce morphological or functional differentiation in SH-SY5Y cells (Jalava et al., 1990). Incubation of SH-SY5Y cells in the presence of 100 n M bryostatin 1 for 24 hours caused a complete disappearance of all irnrnunoreactive a-, p-, and 5-PKC. The level of E-PKC decreased by 70%. Staurosporine induced a partial translocation of the &-isoenzyme but it failed to cause down-regulation of E-PKC. Bryostatin 1-treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation of DNA synthesis, and GAP-43 and NPY mRNA expression. The ability of staurosporine to stimulate tyrosine hydroxylase expression and to increase cellular content of noradrenaline was also unaffected. Taken together the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH-SY5Y cells through an a-, p-, and 5-PKC-independent pathway. o 1993 WiIey-Liss, Inc. SH-SY5Y human neuroblastoma cells are induced t o differentiate t o m a t u r e n e u r o n a l cells w h e n treated with tumour-promoting p h o r b o l esters such as TPA (for a review, see PBhlman e t al., 1990). Characteristic t o TPA-treated S H-S Y 5 Y cells i s an appearance o f long, n e u r i t e l i k e processes with varicosities and g r o w t h cones (PBhlman e t al., 1981), an increased expression o f mRNA for axon-specific g r o w t h associated p r o t e i n (GAP-43) (Bjelfman e t al., 1990), elevated concentrations o f noradrenaline and neuron-specific enolase (Pbhlman e t al., 1981), and an appearance of voltage gated Ca2+ channels (H e i k k i l a e t al., 1989). S H-S Y 5 Y 0 1993 WILEY-LISS, INC cell differentiation i s also associated with a down-regul a t i o n o f c-myc proto-oncogene mRNA (H a m m e r l i n g e t al., 1987; Jalava et al., 1988) and p r o t e i n expression (Jalava e t al., 1992) as w e l l as an increased pp6OC-"'" kinase a c t i v i t y (Bjelfman e t al., 1990).