Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines.

A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.

Human neural tissues express a truncated Ror1 receptor tyrosine kinase, lacking both extracellular and transmembrane domains.

Human heart, lung and kidney express a 6 kb mRNA encoding Ror1, a member of the receptor tyrosine kinase (RTK) family with as yet unknown ligand specificity. We used a Ror1 cDNA probe to screen a cDNA library prepared from the human neuronogenic teratocarcinoma line, NTera2, and cloned a 2373 nucleotide transcript. This transcript contains an open reading frame that encodes a 388 amino acid protein identical with the cytosolic, C-terminal region of ror1 but lacking the ror1 transmembrane and entire extracellular domains. Northern blots demonstrate that mRNA encoding this truncated Ror1 ('t-Rorl') is abundantly expressed in fetal and adult human CNS, in human leukemia, lymphoma cell lines, and in a variety of human cancers derived from neuroectoderm. While previous studies have documented alternative splicing patterns within 5' and 3' regions of mRNAs encoding various RTKs altering their ligand binding specificity or their intracellular signaling, the present report is the first to demonstrate tissue-specific alternative mRNA splicing causing loss of the entire extracellular and transmembrane regions of an RTK.

ZPK inhibits PKA induced transcriptional activation by CREB and blocks retinoic acid induced neuronal differentiation.

Zipper Protein Kinase (ZPK) is a leucine zipper protein localized to the nucleus which exhibits serine-threonine kinase activity and is associated with the stress dependent signal transduction pathway. ZPK forms heterodimers with leucine zipper containing transcription factors such as the cyclic AMP responsive element binding protein (CREB) and Myc. Furthermore ZPK phosphorylates both Myc and CREB. Overexpression of ZPK in NTera-2 human teratocarcinoma cells results in inhibition of PKA induced transcriptional activation by CREB and prevents retinoic acid induced differentiation of the cells to neurons. Our results suggest that ZPK stifles neural differentiation of NT-2 cells partly due to its inhibitory effect on CREB function.

Transient expression of full-length and truncated forms of the human nerve growth factor receptor.

To facilitate the characterization of nerve growth factor (NGF) receptor and mutated forms of the receptor, we have set up a rapid, efficient transient expression system utilizing COS cells. The human NGF receptor is a 427 amino acid protein with a hydrophobic signal sequence, a 222 amino acid extracellular domain, a single transmembrane domain and a 155 amino acid intracellular domain. The NGF receptor and a truncated form lacking the cytoplasmic domain were expressed in a COS cell expression system. Both recombinant proteins were detected on the cell surface and at a perinuclear site. Specific binding of 125I-NGF to the recombinant proteins was detected by chemical cross-linking. The extracellular domain of the NGF receptor was also expressed in the same system and detected in the COS cell endoplasmic reticulum and in the culture supernatant. This recombinant protein also specifically binds NGF.

Expression of platelet-derived growth factor (PDGF) and PDGF receptor genes in the developing rat brain.

Steady-state levels of rat central nervous system (CNS) platelet-derived growth factor (PDGF) A- and B-chain mRNAs were measured by a polymerase chain reaction method employing a synthetic gene internal standard, and the rates of transcription of PDGF A- and B-chain genes in CNS were estimated by a nuclear runoff assay. The abundance of PDGF B-chain mRNA was an order of magnitude below that of PDGF A-chain mRNA, while the rate of PDGF B-chain transcription was only slightly below that for the PDGF A-chain gene, indicating that the half-life of PDGF B-chain in CNS is shorter than that of PDGF A-chain mRNA. No developmental alterations in expression of the PDGF A- and B-chain genes were detected. By contrast, Northern blots showed that steady-state levels of mRNAs encoding the two PDGF receptor proteins, alpha and beta, were markedly higher in embryonic day 15 and postnatal day 6 rat brains than in later life. These results suggest that the actions of PDGF on the brain in vivo are regulated not at the level of PDGF A and B-chain gene expression, but rather by changes in the level of expression of PDGF alpha- and beta-receptor genes.

Cloning of a novel putative protein kinase having a leucine zipper domain from human brain.

We report a novel putative serine/threonine protein kinase containing a leucine-zipper domain, isolated from an human neuronal cell line. The teratocarcinoma cell line NT2 was differentiated to postmitotic NT2-N neurons by treatment with retinoic acid, and degenerate oligonucleotide primers to the catalytic domains of protein kinases were employed to PCR amplify subtractive cDNAs. We identified a clone, represented at higher abundance in NT2-N neurons than in the parental cell line, which encodes a putative serine/threonine kinase of 859 [corrected] amino acids, the leucine-zipper protein kinase (zpk). Zpk protein contains a leucine-zipper domain, found in many DNA-binding proteins, but few protein kinases. Steady-state mRNA levels for zpk are high in human brain and kidney. Further studies are required to evaluate the role of zpk in neuronal differentiation.

Platelet-derived growth factor and regulation of Schwann cell proliferation in vivo.

To examine the role of platelet-derived growth factor (PDGF) in the in vivo regulation of Schwann cell proliferation, steady-state levels of mRNAs encoding PDGF A and B chains, and PDGF alpha and beta receptors were measured in immature and adult rat sciatic nerves and in cultured rat Schwann cells. PDGF B chain and PDGF beta receptor mRNAs are present in immature rat sciatic nerves and to a lesser extent in adult rat nerves. Short-term cultures of neonatal rat Schwann cells express PDGF beta receptor mRNA, but not PDGF B chain mRNA, and are stimulated to synthesize DNA by addition of PDGF BB to the medium. These data indicate that PDGF BB is a developmentally regulated paracrine growth factor for rat Schwann cells. Very long-term cultures of rat Schwann cells, which have lost normal dependence on exogenous growth factors, express PDGF B chain mRNA as well as mRNAs encoding the PDGF alpha and beta receptors, suggesting that, under these circumstances, PDGF BB also act as an autocrine growth factor. PDGF A chain mRNA is present in both immature and adult rat sciatic nerves and is expressed by primary and secondary cultures of rat Schwann cells as well. However, because the abundance of PDGF alpha receptor mRNA is very low in rat Schwann cells, PDGF AA is not likely to be a significant autocrine growth factor for rat Schwann cells.

Diminished calcium homeostasis and increased susceptibility to excitotoxicity of JS 3/16 progenitor cells after differentiation to oligodendroglia.

JS 3/16, derived from passaged oligodendroglial cultures prepared from rat cerebral white matter, differentiate from progenitors (OP) into complex process-bearing, galactocerebroside-positive but myelin basic protein-negative immature oligodendrocyte-like cells (ImO) after withdrawal of trophic factors. We found that JS 3/16 ImO are markedly more susceptible than OP to cell death after sustained alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-GluR) activation. This excitotoxicity is preceded by loss of intracellular Ca(2+) homeostasis, which is more marked in ImO than OP. We identified three factors likely to contribute to the diminished Ca(2+) homeostatic capacity of ImO. First, signal intensities of immunoreactive GluR2, GluR3, and GluR4 AMPA-GluR subunits are increased 1.3- to 2.2-fold in ImO over OP without comparable changes in RNA editing and alternative splicing. Second, transcriptional levels of genes encoding Na(+)-Ca(2+) exchanger proteins and a plasma membrane ATPase (PMCA1), which are necessary for Ca(2+) extrusion across the plasma membrane, are lower in ImO than in OP. Third, ImO have more depolarized basal mitochondrial membrane potential (Delta Psi) than OP, and Delta Psi collapses within 15 min after onset of AMPA-GluR activation in almost all ImO, but not in the majority of OP. This Delta Psi collapse limits the capacity of ImO mitochondria to buffer the rise in intracellular Ca(2+) caused by AMPA-GluR activation. The JS 3/16 line provides a valuable system for analysis of intracellular Ca(2+) homeostasis and AMPA-GluR-mediated excitotoxicity in the oligodendroglial lineage.

Caspase-3 expression by cerebellar granule neurons is regulated by calcium and cyclic AMP.

Caspase-3 enzyme activity is induced, and cell death follows, when cerebellar granule neurons (CGNs) from 8-day-old rats are transferred from an extracellular concentration of 25 mM K+ (25 mM [K+]e) to 5 mM [K+]e. Death of these neurons is diminished by an inhibitor of caspase-3 but not by an inhibitor of caspase-1. Actinomycin D and cycloheximide inhibit induction of caspase-3 and prevent death. Experiments in which CGN intracellular Ca2+ concentration ([Ca2+]i) was manipulated by either changing [K+]e or adding a voltage-gated Ca2+ channel antagonist or a Ca2+ ionophore to the medium showed that caspase-3 mRNA rises 2.5-fold when [Ca2+]i is diminished from 300 to 150 nM, with a corresponding rise in peak caspase enzyme activity. Whereas the caspase-3 mRNA level does not rise further with a still greater diminution in [Ca2+]i, peak caspase enzyme activity continues to increase, reaching sevenfold induction when [Ca2+]i is reduced to 55 nM. In CGNs in which [Ca2+]i is set at 55 nM by incubation in 5 mM [K+]e, treatment with forskolin or dibutyryl 3',5'-cyclic adenosine-5'-monophosphate delays caspase-3 induction and diminishes death but does not alter [Ca2+]i. We conclude that, in immature CGNs, both caspase-3 transcription and the subsequent processing of caspase-3 are induced by a fall in [Ca2+]i. Elevating cyclic AMP content delays caspase-3 induction by a mechanism that does not require an increase in [Ca2+]i.

Axon-Schwann cell interactions regulate the expression of fibroblast growth factor-5 (FGF-5).

We screened for genes whose expression is significantly up- or downregulated during Wallerian degeneration in adult rat sciatic nerve with cDNA arrays. Fibroblast growth factor-5 (FGF-5) mRNA seemed to be induced. This was confirmed by northern blotting and in situ hybridization, as well as Western blotting for FGF-5 in axotomized nerve. Axon-Schwann cell interactions decreased the steady-state level of FGF-5 mRNA in regenerating sciatic nerves, and forskolin diminished its expression in cultured Schwann cells. We conclude that denervated Schwann cells synthesize FGF-5, which is a secreted, neuronotrophic member of the FGF family.

Regulation of Schwann cell nerve growth factor receptor by cyclic adenosine 3',5'-monophosphate.

Previous studies indicated that Schwann cells in immature nerves express nerve growth factor (NGF) receptors, and that this expression is down regulated during development but re-induced by Wallerian degeneration. It was also shown that immature Schwann cells are induced to express galactocerebroside and other molecules characteristic of mature Schwann cells by either contact with an axon or treatment with the cyclic adenosine 3',5'-monophosphate (cAMP) analogues dibutyryl cAMP (dbcAMP) and 8-bromo cAMP or the adenylate cyclase activator forskolin. In the present study, NGF receptors on the surface of cultured Schwann cells were demonstrated by binding of an anti-rat NGF receptor monoclonal antibody or of radioiodinated NGF. Treatment of cultured Schwann cells with cAMP analogues or forskolin resulted in a progressive decrease in both immunoreactive NGF receptors and radioiodinated NGF binding. The cultured Schwann cells contained a polyadenylated RNA species homologous with human melanoma NGF receptor mRNA in sequence and size. The amount of this NGF mRNA was lower in cAMP analogue-treated than in untreated Schwann cells.

Characterization of two neuroblastoma cell lines expressing recombinant nerve growth factor receptors.

In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LAN5 is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Localization of the human Ror1 gene (NTRKR1) to chromosome 1p31-p32 by fluorescence in situ hybridization and somatic cell hybrid analysis.

Ror1 is an orphan cell surface receptor with strong homology to the tyrosine kinase domain of growth factor receptors, in particular the Trk family. Southern blot analysis of genomic DNA from somatic cell hybrids revealed that Ror1 is located on chromosome 1. We have mapped the Ror1 gene to chromosome 1p12-p32 using PCR on a somatic cell hybrid panel that subdivides chromosome 1p. We have further localized the gene to chromosome 1p31-p32 by fluorescence in situ hybridization using a PAC clone that contains the Ror1 gene.

Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.

Human central nervous system primitive neuroectodermal tumor expressing nerve growth factor receptors: CHP707m.

A primitive neuroectodermal tumor (PNET) presented as a cerebral hemispheric mass in a 33-year-old man. Bone marrow metastases were discovered 11 months later. A cell line (CHP707m) was derived from these metastases. In culture, the cells showed features of neuronal differentiation, forming short neurites and synthesizing low-molecular-weight neurofilament protein. Northern blotting showed the tumor cells express nerve growth factor (NGF) receptor messenger RNA, and fluorescence-activated cell-sorting demonstrated NGF receptors on the cell surface. Western blotting showed CHP707m NGF receptors are truncated. The receptors are functional; they bind iodine 125-labeled mouse NGF with an affinity of 1.6 x 10(-9) M, and short-term treatment with NGF induces expression by the tumor cells of the proto-oncogene, c-fos. Although CHP707m is the first central nervous system PNET cell line proven to express NGF receptors, immunohistological survey of tissue sections prepared from human central nervous system PNETs showed that 13 of 35 contained NGF receptor-positive tumor cells. Thus, more than one-third of such tumors might be responsive to the effects of NGF.

Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.