Mark A. Marchionni

Glial growth factor restricts mammalian neural crest stem cells to a glial fate

Growth factors and cytokines are thought to influence the development of uncommitted progenitor cell populations, but the issue of how these factors act on individual cells remains controversial. Such factors may act simply as selective mitogens or survival factors for cells that undergo lineage restrictions stochastically. Alternatively, they may instruct or bias multipotent cells to choose one lineage at the expense of others. Here we show that glial growth factor (GGF), previously defined as a Schwann cell mitogen, strongly suppresses neuronal differentiation of rat neural crest stem cells while promoting or allowing glial differentiation. Quantitative clonal analysis suggests that the action of GGF is likely to be instructive rather than selective. Taken together with the expression pattern of GGF, these data suggest a lateral signaling model for the diversification of cell types within developing peripheral ganglia.

Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells

Schwannomas are peripheral nerve tumors that typically have mutations in the NF2 tumor suppressor gene. We compared cultured schwannoma cells with Schwann cells from normal human peripheral nerves (NHSC). Both cell types expressed specific antigenic markers, interacted with neurons, and proliferated in response to glial growth factor, confirming their identity as Schwann cells. Schwannoma cells frequently had elevated basal proliferation compared to NHSC. Schwannoma cells also showed spread areas 5–7-fold greater than NHSC, aberrant membrane ruffling and numerous, frequently disorganized stress fibers. Dominant negative Rac inhibited schwannoma cell ruffling but had no apparent effect on NHSC. Schwannoma cell stress fibers were inhibited by C3 transferase, tyrphostin A25, or dominant negative RhoA. These data suggest that the Rho and Rac pathways are abnormally activated in schwannoma cells. Levels of ezrin and moesin, proteins related to the NF2 gene product, merlin, were unchanged in schwannoma cells compared to NHSC. Our findings demonstrate for the first time that cell proliferation and actin organization are aberrant in schwannoma cells. Because NF2 is mutant in most or all human schwannomas, we postulate that loss of NF2 contributes to the cell growth and cytoskeletal dysfunction reported here.

Schwann Cells Express NDF and SMDF/n-ARIA mRNAs, Secrete Neuregulin, and Show Constitutive Activation of erbB3 Receptors: Evidence for a Neuregulin Autocrine Loop

Cultured Schwann cells secreted low levels (30 pg/ml/1.5 x 10(6) cells) of a 45-kDa neuregulin protein and showed constitutive activation of a neuregulin receptor, Erb-B3, suggesting the existence of an autocrine loop involving neuregulins in Schwann cells. RT-PCR analyses indicated that Schwann cells and fibroblasts in culture produced SMDF/n-ARIA and NDF but not GGF neuregulin messages. Schwann cell and fibroblast neuregulin messages encoded both beta and alpha domains; Schwann cell transcripts encoded only transmembrane neuregulin forms while fibroblast messages encoded transmembrane and secreted forms. SMDF/n-ARIA and NDF messages were also expressed in early postnatal rat sciatic nerve, suggesting a role for neuregulins in peripheral nerve development. An anti-neuregulin antibody inhibited the mitogenic response of Schwann cells to cultured neurons and to extracts of cultured neurons or embryonic brain, consistent with the accepted paracrine role of neuregulins on Schwann cells. Surprisingly, the same antibody inhibited Schwann cell proliferation stimulated by several unrelated mitogens including bFGF, HGF, and TGF-beta1. These data implicate both paracrine and autocrine pathways involving neuregulin form(s) in Schwann cell mitogenic responses.

Neuregulin in Neuron/Glial Interactions in the Central Nervous System

Glial growth factor 2 (GGF2) is a neuronal signal that promotes the proliferation and survival of the oligodendrocyte, the myelinating cell of the central nervous system (CNS). This study has focused on recombinant human GGF2 (rhGGF2) and its potential to affect clinical recovery and repair to damaged myelin in chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the mouse, a major animal model for the human demyelinating disease, multiple sclerosis (MS). Mice with EAE were treated with rhGGF2 during both the acute and relapsing phases, and GGF2 treatment led to delayed signs, decreased severity and resulted in statistically significant reductions in relapse rate. Further, rhGGF2-treated groups displayed CNS lesions with more remyelination than in controls. This correlated with increased expression of myelin basic protein exon 2, a marker for remyelination, and with an increase of the regulatory cytokine, IL-10. Thus, a beneficial effect of a neurotrophic growth factor has been demonstrated upon the clinical, pathologic and molecular manifestations of autoimmune demyelination, an effect that was associated with increased expression of a Th2 cytokine. rhGGF2 treatment may represent a novel approach to the treatment of MS (Cannella et al., 1998).

Replication of viral DNA sequences integrated within the chromatin of SV40-transformed chinese hamster lung cells

To determine when during S phase integrated viral DNA sequences in several tsA SV40-transformed Chinese hamster cell clones replicate, we pulse-labeled cultures with BrdUrd and subsequently collected mitotic cells during sequential time intervals. Restriction endonuclease mapping indicated that each of the three SV40-transformed Chinese hamster lung cell clones contained a single viral DNA sequence at a different, but in each case unique, chromosomal site. DNA was extracted from each population of mitotic cells and then was resolved into dense, BrdUrd-containing and light, unsubstituted DNA fractions by cesium chloride gradient centrifugation. In each pair of samples obtained, we measured viral DNA sequences by solution hybridization using single-stranded SV40 32P-labeled DNA probes. Our results support the conclusions that specific genes within a mammalian DNA are programmed to replicate at particular times during S phase, and that the SV40 A gene product, large T antigen, programs integrated viral DNA sequences to replicate very early in S phase. The fractions of viral DNA replicated early in S phase appeared to correlate with each clones content of functional large T antigen at permissive and nonpermissive culture temperatures.