George H. DeVries

Ral overactivation in malignant peripheral nerve sheath tumors.

ABSTRACT Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.

N-acetylneuraminic acid aldolase of Clostridium perfringens: Purification, properties and mechanism of action

Abstract A procedure for purifying N -acetylneuraminic acid aldolase of Clostridium perfringens is described. The purified enzyme has a molecular weight of 92,000 and consists of two protein components each of which has enzymatic activity. The purified aldolase has a pH optimum of 7.2 and a K m of 1.75 m m ; heavy metal ions are potent inhibitors. No metal ion requirement could be demonstrated for the enzyme and it is completely inhibited by sodium borohydride reduction in the presence of N -acetylneuraminic acid. This indicates that the enzyme is a Class I aldolase and forms a Schiff base-enzyme complex. There is no requirement for a substrate carboxyl group for binding to occur; substitution of a polar or bulky group at the deoxy position causes decreased binding. Substrate analog binding did not result in effective cleavage of the appropriate carbon-carbon bond. Kinetic studies with the pyruvate analog bromopyruvate are consistent with the existence of nucleophilic residues within the active site.

Schwann Cell Lines Derived From Malignant Peripheral Nerve Sheath Tumors Respond Abnormally to Platelet-Derived Growth Factor-BB

Neurofibromatosis type 1 (NF1) is a genetic disease caused by the loss of neurofibromin, which can lead to formation of highly invasive malignant peripheral nerve sheath tumors (MPNST). We characterized platelet‐derived growth factor‐β (PDGF‐β) receptor expression levels and signal transduction pathways in NF1 MPNST cell lines and compared them with the expression of PDGF‐β receptors in normal human Schwann cells (nhSC). As examined by Western blotting, PDGF‐β receptor expression levels were similar in nhSC and NF1 MPNST cell lines. MAPK and Akt also were phosphorylated in both cell types to a similar degree in response to PDGF B chains (PDGF‐BB). However, increased intracellular calcium (Ca2+) levels in response to PDGF‐BB were observed only in the NF1 MPNST cell lines; nhSC did not show any increase in intracellular calcium when stimulated with PDGF‐BB. The calcium response in NF1 MPNST cell lines was blocked with thapsigargin, suggesting that the PDGF‐BB‐stimulated increases in intracellular calcium originated in the internal compartment of the cell rather than reflecting influx of calcium from the extracellular compartment. Calmodulin kinase II (CAMKII) is phosphorylated in response to PDGF‐BB in the NF1 MPNST cell lines, whereas no phosphorylation of CAMKII was observed in nhSCs. The decreased growth of NF1 MPNST cell lines after treatment with a CAMKII inhibitor is consistent with the view that aberrant activation of the calcium‐signaling pathway by PDGF‐BB contributes to the formation of MPNST in NF1 patients.

Plexiform‐like neurofibromas develop in the mouse by intraneural xenograft of an NF1 tumor‐derived Schwann cell line

Plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1 (NF1) and have a risk of malignant progression. Past efforts to establish xenograft models for neurofibroma involved the implantation of tumor fragments or heterogeneous primary cultures, which rarely achieved significant tumor growth. We report a practical and reproducible animal model of plexiform‐like neurofibroma by xenograft of an immortal human NF1 tumor‐derived Schwann cell line into the peripheral nerve of scid mice. The S100 and p75 positive sNF94.3 cell line was shown to possess a normal karyotype and have apparent full‐length neurofibromin by Western blot. These cells were shown to have a constitutional NF1 microdeletion and elevated Ras‐GTP activity, however, suggesting loss of normal neurofibromin function. Localized intraneural injection of the cell line sNF94.3 produced consistent and slow growing tumors that infiltrated and disrupted the host nerve. The xenograft tumors resembled plexiform neurofibromas with a low rate of proliferation, abundant extracellular matrix (hypocellularity), basal laminae, high vascularity, and mast cell infiltration. The histologic features of the developed tumors were particularly consistent with those of human plexiform neurofibroma as well. Intraneural xenograft of sNF94.3 cells enables the precise initiation of intraneural, plexiform‐like tumors and provides a highly reproducible model for the study of plexiform neurofibroma tumorigenesis. This model facilitates testing of potential therapeutic interventions, including angiogenesis inhibitors, in a relevant cellular environment.

c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors.

The growth factor receptor c‐Kit has several well‐characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [ 1998 ] Oncogene 17:795–800). However, c‐Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c‐Kit levels, whereas expression levels for c‐Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c‐Kit expression is not detectable in normal adult human Schwann cells. Although the c‐Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells.

3-Hydroxy-N-acetylneuraminic acid: Synthesis and inhibitory properties

Abstract 3-Hydroxy-N-acetylneuraminic acid was synthesized by the condensation of N-acetyl d -mannosamine with bromopyruvate or hydroxypyruvate in yields of 24.4 and 12.7%, respectively. The product was characterized by its infrared and nuclear magnetic resonance spectra, thin-layer chromatography, optical rotation, neutralization equivalent and direct Ehrlich chromogen. This substituted sialic acid was a weak noncompetitive inhibitor of N-acetyl neuraminic acid aldolase purified from Clostridium perfringens. The inhibitor had a Ki of 75 m m and a minimum inactivation halftime of 0.80 min. Hydroxypyruvate was an effective noncompetitive inhibitor of the aldolase with a Ki of 0.70 m m . The mechanism of inhibition and possible reasons for the difference between the effectiveness of hydroxypyruvate and 3-hydroxy-N acetylneuraminic acid as noncompetitive inhibitors are discussed.

Developmental Regulation of Neuregulin1 Isoforms and erbB Receptor Expression in Intact Rat Dorsal Root Ganglia

Neuregulins (NRGs) are a family of growth factors which bind to the erbB family of tyrosine kinase receptors. The exact nature and interaction of specific NRG isoforms and erbB receptors that occur during the development of the nervous system have not been reported. In order to better understand the role that different NRG isoforms and erbB receptors play in the differentiation, proliferation, and survival of neurons and glial cells, we isolated protein and mRNA from dorsal root ganglia of rat pups between embryonic day (E) 13 and postnatal day (P) 15. The relative expression levels of the NRGs and erbB receptors for the different time points were compared using both Western and RT-PCR analyses. NRG1-type1α protein levels were highest at E-13 and then decreased by approximately 40% and remained constant through P-15. In contrast, mRNA levels for NRG1-type1α remained constant from E-15 to P-15. The protein levels for NRG1-type 1β were similar to NRG1-type1α at E-13 with an approximate 40% increase in the levels at E-15 and E-17 followed by a decrease to E-13 levels for the remainder of the developmental time periods. The mRNA levels for NRG1-type1β remained constant from E-15 to P-15. The protein and mRNA expression patterns for each erbB receptor were distinctive. The protein levels for erbB-2 were highest at E-19 while erbB-3 levels were highest at E-17 and E-18. ErbB-4 protein levels were highest at E-13 and decreased through P-15. The developmental pattern for erbB-2 and erbB-4 mRNA levels had no relation to that of the corresponding protein levels while the mRNA levels for erbB-3 were highest at E-17 and E-18 similar to the pattern observed for the erbB-3 protein levels. We concluded that both NRG and erbB expression in dorsal root ganglia are mostly translationally controlled and that NRG1 isoforms and their erbB receptors are not coordinately regulated.

Differential localization of neuregulin-1 type III in the central and peripheral nervous system

In the developing PNS, axonal neuregulin-1 (NRG1) type III is the key determinant for myelination. However, the specific role for NRG1 (III) in the CNS has not been established. To address this issue, isotype-specific antibodies were generated, characterized, and used for the immunofluorescent localization of NRG1 (III) in the developing and adult CNS of rat. In contrast to adult peripheral nerve, which showed robust axonal staining, no immunoreactivity was observed in CNS myelinated tracts during the period of active myelination or in the adult CNS. Surprisingly, NRG1 (III) was prominently expressed on dendrites and soma in both the developing and adult CNS. These findings were corroborated through the subcellular fractionation of adult rat brain combined with an immunoblotting analysis. The immunolocalization of NRG1 (III) suggests that it plays a novel role in the myelination fate of CNS axons possibly through undetermined roles in neuronal maturation, or dendritic development and activation.

Oligodendrocyte progenitor cells proliferate and survive in an immature state following treatment with an axolemma-enriched fraction

The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.

Schwann cells express IP prostanoid receptors coupled to an elevation in intracellular cyclic AMP.

We have shown previously that prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are each produced in an explant model of peripheral nerve injury. We report that IP prostanoid receptor mRNA and protein are present in primary rat Schwann cells. IP prostanoid receptor stimulation using prostacyclin produced an elevation in intracellular cyclic AMP concentration ([cAMP]i) in primary Schwann cells. Peak [cAMP]i was observed between 5–15 min of stimulation followed by a gradual recovery toward basal level. Phosphorylation of cyclic AMP‐response element binding protein (CREB) on Ser133 was also detected after IP prostanoid receptor stimulation and CREB phosphorylation was inhibited completely by the protein kinase A inhibitor, H‐89. Intracellular calcium levels were not affected by IP prostanoid receptor stimulation. Unlike forskolin, IP prostanoid receptor stimulation did not significantly augment Schwann cell proliferation in response to growth factor treatment. However, IP prostanoid receptor stimulation increased the number of Schwann cells that were able to generate a calcium transient in response to P2 purinergic receptor activation. These findings suggest that signaling via the IP prostanoid receptor may by relevant to Schwann cell biology in vivo.