| 1. |
- Fagerström, Sofia, et al.
(författare)
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Protein kinase C-dependent tyrosine phosphorylation of p130cas in differentiating neuroblastoma cells
- 1998
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 273:4, s. 2336-2343
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Tidskriftsartikel (refereegranskat)abstract
- The cell signaling docking protein p130cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c-src. The TPA-induced p130cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-epsilon induced p130cas tyrosine phosphorylation. pp60c-src, known to directly phosphorylate p130cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130cas. Also, overexpression of src induced phosphorylation of p130cas. p130cas protein and phosphorylated p130cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130cas. These data suggest a function for p130cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.
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| 2. |
- Fagerström, Sofia, et al.
(författare)
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Protein kinase C-epsilon is implicated in neurite outgrowth in differentiating human neuroblastoma cells
- 1996
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Ingår i: Cell growth & differentiation. - : American Association of Cancer Research. - 1044-9523. ; 7:6, s. 775-785
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Tidskriftsartikel (refereegranskat)abstract
- A combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) or 16 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum induces human SH-SY5Y neuroblastoma cells to undergo differentiation and acquire a neuronal phenotype. Nerve growth factor (NGF) added to SH-SY5Y cells stably transfected with the NGF-receptor TRK-A (SH-SY5Y/trk) induces a similar differentiated phenotype. SH-SY5Y cells express protein kinase C (PKC)-alpha, PKC-beta I, PKC-epsilon, and PKC-zeta protein, and phorbol ester- or growth factor-induced differentiation results in a sustained activation of PKC. The specific PKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite outgrowth in wild-type SH-SY5Y cells and NGF-induced neurite outgrowth in SH-SY5Y/trk cells. When added to differentiated cells, GF 109203X caused rapid retraction of growth cone filopodia. In TPA- and bFGF-IGF-I-treated cells, addition of GF 109203X also blocked induced expression of growth associated protein-43 and neuropeptide tyrosine while the increase in expression of these two genes was only slightly affected by the inhibitor in NGF-treated SH-SY5Y/trk cells. Thus, a portion of the NGF-induced phenotypic changes appears not to be mediated via PKC-dependent signaling. A high concentration of TPA (1.6 microM) down regulated PKC-alpha and PKC-beta I almost completely and PKC-epsilon partially in wild-type SH-SY5Y and SH-SY5Y/trk cells. Cells with down-regulated PKC-alpha and PKC-beta I after 1.6 microM TPA treatment still differentiated with growth factors. In these cells, the PKC-epsilon level was restored, and the PKC-epsilon protein was enriched in the growth cones. The 1.6 microM TPA-induced down-regulation of PKC-epsilon was counteracted by bFGF and NGF but not by platelet-derived growth factor or IGF-I. These data indicate that PKC activity is vital for neurite formation, and that the cells can differentiate under conditions when PKC-alpha and PKC-beta I are extensively down regulated. The close correlation between differentiation and presence of PKC-epsilon protein suggests an important function for this isoform during this process.
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| 3. |
- Gestblom, C, et al.
(författare)
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The basic helix-loop-helix transcription factor dHAND, a marker gene for the developing human sympathetic nervous system, is expressed in both high- and low-stage neuroblastomas
- 1999
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Ingår i: Laboratory Investigation. - 1530-0307 .- 0023-6837. ; 79, s. 67-
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Tidskriftsartikel (refereegranskat)abstract
- Neuroblastoma is derived from the sympathetic nervous system and might arise as a result of impaired differentiation, retaining the neuroblastic tumor cells in the cell cycle. Thus, to understand the genesis of neuroblastoma, the study of mechanisms and genes regulating normal sympathetic development is of potential interest. The basic helix-loop-helix transcription factors human achaete-scute homolog-1 (HASH-1) and deciduum, heart, autonomic nervous system, and neural crest derivatives (dHAND) are expressed in the sympathetic nervous system of embryonic mice and chicken, with undetectable postnatal expression. By in situ hybridization technique, we show that dHAND was expressed by human sympathetic neuronal and extra-adrenal chromaffin cells throughout embryonic and fetal life, and was initially expressed in immature chromaffin cells of the adrenal gland. With overt chromaffin differentiation, dHAND was down-regulated. HASH-1, in contrast, was expressed in human sympathetic cells only at the earliest embryonic ages examined (Week 6.5 to 7). All examined neuroblastoma specimens (25/25) and all cell lines (5/5) had detectable dHAND mRNA levels. HASH-1 expression in tumor specimens was more restricted, although all cell lines (5/5) were HASH-1-positive. These results show that neuroblastoma tumors have retained embryonic features, suggesting that many neuroblastomas are blocked at an early stage of normal development when HASH-1 and dHAND are expressed. dHAND also appears to be a reliable and potentially useful clinical diagnostic marker for neuroblastoma, because expression was not dependent on tumor or differentiation stages and other pediatric tumors were dHAND-negative.
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| 4. |
- Lavenius, Erik, et al.
(författare)
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Transfection of TRK-A into human neuroblastoma cells restores their ability to differentiate in response to nerve growth factor
- 1995
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Ingår i: Cell growth & differentiation. - : American Association of Cancer Research. - 1044-9523. ; 6, s. 727-736
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Tidskriftsartikel (refereegranskat)abstract
- Human neuroblastoma cell lines frequently express the TRK-A proto-oncogene and bind nerve growth factor (NGF) but do not differentiate when exposed to NGF. Transient transfection of an exogenous TRK-A gene into SH-SY5Y and LA-N-5 neuroblastoma cells restored the ability of these tumor cells to differentiate with NGF. Stable TRK-A-transfected SH-SY5Y cell clones were isolated, and they responded to NGF by autophosphorylation of p140trk-A, c-fos induction, morphological differentiation, and increased expression of two neuronal marker genes, neuropeptide tyrosine and GAP-43. In phorbol ester-induced differentiated wild-type cells, TRK-A expression was increased with no change in NGF responsiveness. Thus, the restoration of the NGF-induced differentiation pathway by exogenous TRK-A presents a system of NGF-responsive human cultured cells and focuses attention on the trk-A protein and its function or malfunction in neuroblastoma.
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| 5. |
- Söderholm, Helena, 1969-, et al.
(författare)
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Human achaete-scute homologue 1 (HASH-1) is downregulated in differentiating neuroblastoma cells
- 1999
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 256:3, s. 557-563
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian achaete-scute homologue, MASH-1, is crucial for early development of the sympathetic nervous system and is transiently expressed in sympathetic neuroblasts during embryogenesis. Here we report that the human homologue (HASH-1) was expressed in all analyzed cell lines (6/6) derived from the sympathetic nervous system tumor neuroblastoma. The majority of small-cell lung carcinoma (4/5) cell lines tested expressed HASH-1, while other nonneuronal/non-neuroendocrine cell lines were negative. Induced differentiation of neuroblastoma cells resulted in HASH-1 downregulation. This occurred concomitant with induction of neurite outgrowth and expression of the neuronal marker genes GAP-43 and neuropeptide Y. Constitutive expression of exogenous HASH-1 did not alter the capacity of the neuroblastoma cells to differentiate in response to differentiation-inducing agents. It is concluded that moderate HASH-1 expression does not compromise the capacity of these cells to differentiate.
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| 6. |
- Zeidman, Ruth, et al.
(författare)
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Novel and classical protein kinase C isoforms have different functions in proliferation, survival and differentiation of neuroblastoma cells
- 1999
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Ingår i: International Journal of Cancer. - 0020-7136. ; 81:3, s. 494-501
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Tidskriftsartikel (refereegranskat)abstract
- To elucidate the possibility of utilizing protein kinase C (PKC) isoforms as target genes in neuroblastoma therapy, 5 neuroblastoma cell lines and neuroblastoma tumor specimens were examined for PKC isoform expression pattern and the cell lines were analyzed for sensitivity to PKC inhibition. All cell lines [IMR-32, LAN-2, LAN-5, SH-SY5Y and SK-N-BE(2)] expressed alpha, betaII, delta and epsilon isoforms of PKC, while no PKCeta or theta protein was detected in any cell line. PKCgamma was found only in LAN-2 cells. PKCalpha, betaII and delta were detected in 5 neuroblastoma tumors and PKCepsilon in 4 out of 5 tumors. Exposure to the PKC inhibitors GF109203X, Go 6976 or Go 6983 caused a decrease whereas activation of PKC with 12-O-tetradecanoyl phorbol 13-acetate caused an increase in the number of neuroblastoma cells. The effect of Go 6976 was due to both inhibited proliferation and to increased apoptosis. While GF109203X suppressed neurite outgrowth induced by a growth factor combination, Go 6976 potentiated neurite outgrowth. Our data suggest a role for classical PKC isoforms in neuroblastoma growth and survival and for novel isoforms in neurite outgrowth.
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| 7. |
- Zeidman, Ruth, et al.
(författare)
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PKCepsilon, via its regulatory domain and independently of its catalytic domain, induces neurite-like processes in neuroblastoma cells
- 1999
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Ingår i: Journal of Cell Biology. - 0021-9525. ; 145:4, s. 713-726
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCalpha, betaII, delta, and epsilon fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCepsilon-EGFP induced cell processes whereas the other isoforms did not. The effect of PKCepsilon-EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCepsilon regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCepsilon and neurites induced during retinoic acid- and growth factor-induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCepsilon- PSC1V3 protein contained alpha-tubulin, neurofilament-160, and F-actin, but the PKCepsilon-PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y. These data suggest that PKCepsilon, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.
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