Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorageindependent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.

After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.

Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HSs) and fibroblast growth factors (FGFs) 1 and 9 as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC-conditioned and rat OECconditioned media showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semisynthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared with OECs, and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs, compared with SCs, to suppress boundary formation. Furthermore, specific anti-FGF1 and anti-FGF9 antibodies disrupted SC-astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signaling mechanism via FGF receptors on astrocytes. We propose a model in which FGF1 and FGF9 signaling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGF receptor 3-IIIb-mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair.

Background: Debate has been ongoing on the relative merits of olfactory ensheathing cells (OECs) and Schwann cells as candidates for transplant-mediate repair of CNS lesions. Both glial cells exhibit similar molecular and cellular properties and to date there has been no antigenic marker identified that can clearly distinguish the two cell types. This inability to distinguish between the two cells types prevents confirmation of a controversial statement that cultures of OECs are contaminated with Schwann cells. Recently, proteomic analysis of foetal OECs and adult Schwann cells identified an actin-binding protein, calponin, as a specific marker for OECs. However, at the same time a recent report suggested that adult OECs do not express calponin. It was not clear if this discrepancy was due to methodology, as cells had to be treated with proteinase K to maximize calponin staining or developmental differences with only foetal/neonatal OECs expressing calponin. For this reason we have examined calponin expression in the peripheral olfactory system of embryonic and neonatal rats in vivo and from cells in vitro to assess if calponin is expressed in a developmental manner.

No abstract

S U M M A R Y Leishmania amazonensis, an obligatory intracellular parasite, survives internalization by macrophages, but no information is available on the involvement of microglia. We have investigated microglia-protozoa interactions in mixed glial cultures infected with promastigote forms of L. amazonensis after lipopolysaccharide (LPS) or dexamethasone (DM) treatment. After 2 hr of exposure to parasites in control cultures, there was a small number of infected microglia (1%). Preincubation with LPS or DM led to 14% or 60% of microglial cells with attached parasites, respectively. DM treatment resulted in 39% of microglial cells with internalized parasites (controls or LPS-treated cells had Յ 1%). Scanning electron micrographs showed numerous filopodia in DM-treated cells, whereas these projections were rarely observed in LPS-treated or control cells. DM treatment also affected the intramicroglial survival of Leishmania . In control cultures, internalized parasites, tagged with an anti-lipophosphoglycan (anti-LPG) antibody, showed fragmented DNA [terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL ϩ )] after 4 hr of interaction, but changes seemed slightly delayed in DM-treated cultures. After 12 hr, there were no LPG ϩ /TUNEL ϩ profiles in controls, whereas rare LPG ϩ profiles still persisted in DM-treated cells. Our results suggest that microglia are highly effective in the elimination of Leishmania and that the process can be effectively studied by LPG/TUNEL double labeling.

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9‐O‐acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti‐9‐O‐acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G‐A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9‐O‐acetyl GD3 and A2B5 antibodies, and that 2′,3′‐cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9‐O‐acetyl GD3 and with G‐A2B5. Additionally, we showed that the immune blockade of 9‐O‐acetyl GD3 or G‐A2B5 reduced the migration of OEG on laminin, and that 9‐O‐acetyl GD3 and G‐A2B5 colocalized with the β1‐integrin subunit. We also confirmed the phenotype of in‐vitro‐grown OEG cells derived from adult rats, showing that they express CNPase, and also α‐smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9‐O‐acetyl GD3 and G‐A2B5 participate in the migratory activity of OEG cells, and that the β1‐integrin subunit colocalizes with these gangliosides. These results suggest a new role for β1‐integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.

Supplementary Table 2 from Ovarian Cancer Stem Cell–Like Side Populations Are Enriched Following Chemotherapy and Overexpress <i>EZH2</i>